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Serum phenylalanine concentrations in patients with ovarian carcinoma correlate with concentrations of immune activation markers and of isoprostane-8
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Cancer Letters 272 (2008) 141–147 www.elsevier.com/locate/canlet
Serum phenylalanine concentrations in patients with ovarian carcinoma correlate with concentrations of immune activation markers and of isoprostane-8 Gabriele Neurauter a, Anna V. Grahmann b, Martin Klieber a, Alain Zeimet c, Maximilian Ledochowski d, Barbara Sperner-Unterweger b, Dietmar Fuchs a,* a
Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz Pregl Strasse 3, 6020 Innsbruck, Austria b Department of Psychiatry, Innsbruck Medical University, Innsbruck, Austria c Department of Gynaecology and Obstetrics, Innsbruck Medical University, Innsbruck, Austria d Department of Internal Medicine, Innsbruck Medical University, Innsbruck, Austria Received 27 May 2008; received in revised form 27 May 2008; accepted 3 July 2008
Abstract Increased blood concentrations of essential amino acid phenylalanine are common in patients with HIV infection, in trauma and sepsis and in patients with cancer. The reason for this phenomenon is still unclear. However, all these clinical conditions are known to be linked with inflammation and immune activation. Oxidative stress resulting from chronic immune activation and inflammation could impair activity of phenylalanine (4)-hydroxylase (PAH) and thus give rise to increased phenylalanine concentrations. We therefore examined in 20 patients with ovarian cancer a possible association of serum concentrations of phenylalanine and tyrosine with immune activation markers 75 kDa soluble tumor necrosis factor-a receptor (sTNF-R75) and neopterin, and of oxidative stress marker isoprostane-8. Phenylalanine concentrations were higher in patients with higher FIGO stage and correlated with concentrations of sTNF-R75 (rs = 0.441) and neopterin (rs = 0.346; both p < 0.05). No such correlations existed for tyrosine levels. The phenylalanine to tyrosine ratio (phe/tyr), an estimate of PAH activity, correlated somewhat stronger with sTNF-R75 (rs = 0.549; p < 0.01) and neopterin (rs = 0.497; p = 0.01). Finally, phenylalanine concentrations correlated with isoprostane-8 concentrations (rs = 0.450, p = 0.02). Correlations of phenylalanine and phe/tyr with immune activation markers point to a potential role of inflammation and immune activation in the accumulation of phenylalanine. The relationship between oxidative stress marker isoprostane-8 and phenylalanine as well as sTNF-R75 concentrations suggests a link between reactive oxygen species formed during chronic immune activation and inflammation and the decline of PAH activity, which might underlie the increase of phe/tyr (248 words). Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Phenylalanine; Tyrosine; Phenylalanine hydroxylase; Oxidative stress; Immune activation
*
Corresponding author. Tel.: +43 152 9003 70350; fax: +43 512 9003 73330. E-mail address: [email protected] (D. Fuchs).
0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.07.002
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1. Introduction Increased blood concentrations of essential amino acid phenylalanine have been described in consuming diseases such as HIV-1 infection, trauma, sepsis, burn, and malignancy [1–6]. The reason for this increase of phenylalanine is unexplained. However, these clinical conditions are known to be linked with inflammation and immune activation and with increased concentrations of immune activation markers such as serum soluble 75 kDa tumor necrosis factor-a (sTNF-R75) [7] and neopterin [8]. Subnormal enzymatic conversion of phenylalanine by phenylalanine-4-hydroxylase (PAH) might explain the accumulation of phenylalanine in patients [9,10]. Phenylalanine is the precursor of tyrosine, an important amino acid for the biosynthesis of neurotransmitters like L-DOPA (L3,4-dihydroxyphenylalanine) and catecholamines dopamine, epinephrine and norepinephrine. For enzymatic hydroxylation of phenylalanine to tyrosine by PAH, the cofactor 5,6,7,8-tetrahydrobiopterin (BH4), the reduced form of biopterin, is needed as a hydrogen donator [9]. TNF-a receptor (75 kDa) is shed from activated immunocompetent cells [7], and also neopterin is a product, which accumulates during states of immune activation, because large amounts of it are released by monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-c (IFN-c) [11,12]. Increased concentrations of sTNF-R75 and neopterin are common in the blood of patients suffering from conditions like virus infections, autoimmune syndromes and malignancy [7,8]. In parallel to neopterin and sTNF-R75 formation, macrophages and other cells stimulated with IFN-c produce reactive oxygen species (ROS) at a high rate [13]. Overwhelming production of ROS may wipe out antioxidant defence systems and, in turn, oxidative stress is developing. PAH cofactor BH4 is extremely sensitive to oxidation [14], and if BH4 is destroyed, the conversion rate of phenylalanine to tyrosine by PAH is reduced, it should be reflected by an increased phenylalanine to tyrosine ratio (phe/tyr), an estimate of PAH [10,15]. Oxidative stress due to chronic immune activation and inflammation could be involved in the increase of serum phenylalanine concentrations in patients. Recently we described a correlation between phe/tyr and neopterin concentrations in patients after multiple trauma [16].
To further substantiate the possible relationship between inflammation and immune activation and disturbed conversion of phenylalanine to tyrosine, we determined serum concentrations of these amino acids in 21 patients with ovarian carcinoma and compared the results with concentrations of inflammation and immune activation markers sTNF-R75 and neopterin and isoprostane-8, a peroxidation product of arachidonic acid, which represents a marker of oxidative stress [17]. 2. Materials and methods 2.1. Patients Twenty female patients (mean age: 53; range 27– 74 years) with ovarian cancer were recruited from the Department of Gynaecology at the University Clinics of Innsbruck, Austria. Five patients presented with FIGO stage Ib–Ic, 2 with FIGO IIa, 12 with FIGO IIIb–IIIc, and 1 with FIGO IV (Table 1). Degree of malignancy was Stage 1 in 3, Stage 2 in 8, and Stage 3 in 9. Twelve patients (61.9%) were postoperative without residual tumor, 8 (38.1%) were with residual tumor, 9 were premenopausal, 1 perimenopausal, and 10 postmenopausal. All participants’ rights were protected, and according to the Helsinki Declaration informed consent was obtained that a small portion of their blood collected for routine examinations was forwarded for further scientific testings. Table 1 Clinical data of 20 patients with ovarian cancer Number Tumor type
Stage
Malignancy grade
Post-operative status Menopausal status
Mucinous cystadenocarcinoma Serous cystadenocarcinoma Endometroid carcinoma Other FIGO Ib FIGO Ic FIGO IIa FIGO IIIb FIGO IIIc FIGO IV Grade 1 Grade 2 Grade 3 Residual tumor No residual tumor Premenopausal Perimenopausal Postmenopausal
3 15 1 1 3 2 1 1 12 1 3 8 9 12 8 9 1 10
G. Neurauter et al. / Cancer Letters 272 (2008) 141–147
2.2. Laboratory examinations Blood serum samples were collected after surgical treatment of patients was performed and before chemotherapy was started. Samples were kept frozen at 20 °C until analysis. Phenylalanine and tyrosine concentrations were determined by high performance liquid chromatography monitoring their natural fluorescence at an excitation wavelength of 210 nm and an emission wavelength of 302 nm simultaneously [18]. Serum (100 ll) was diluted with 100 ll of 500 lM 3-nitro-L-tyrosine (internal standard) and 25 ll of 2 M trichloroacetic acid was used to precipitate and separate proteins. After centrifugation, supernatants of the samples were diluted 1:25 with 0.015 M potassium dihydrogen-phosphate, which was also used as elution buffer on HPLC. In parallel to the sera, an albumin-based calibration mixture was prepared which contained 100 lM phenylalanine and 100 lM tyrosine and underwent the same pre-analytical procedures as serum specimens. To estimate the activity of PAH, the ratio of the substrate phenylalanine versus the concentrations of the enzyme product tyrosine (=phe/tyr) was calculated [10,19]. Concentrations of sTNF-R75 (R&D Systems, Minneapolis, MN), neopterin (BRAHMS Diagnostica, Hennigsdorf/ Berlin, Germany) and isoprostane-8 (IBL, Hamburg, Germany) were measured by commercially available ELISAs. 2.3. Statistical analysis Concentrations of analytes were given as mean values ± SEM. Because some of the data did not show normal distribution, non-parametric Friedman test and Wilcoxon signed ranks test were applied for comparison of grouped data. Associa-
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tions between marker concentrations were calculated using Spearman rank correlation technique. The Statistical Package for the Social Sciences (version 14 SPSS, Chicago, Ill, USA) was used. p-values below 0.05 were considered to indicate significant differences and correlations. 3. Results Phenylalanine concentrations were 90.2 ± 4.38 lM and tyrosine concentrations were 62.2 ± 2.62 lM, phe/ tyr was 1.47 ± 0.071 (Table 1). Phenylalanine concentrations were significantly higher in patients with FIGO stage III and IV as compared to stages I and II (U = 2.154, p < 0.05; Table 2), whereas tyrosine concentrations did not differ between the two groups. Also phe/tyr was higher in FIGO stages III and IV than in patients with FIGO stage I and II, but the difference was only of borderline significance (U = 1,78, p = 0.075). There were significant correlations between FIGO stage and phenylalanine concentrations (rs = 0.523, p = 0.01) and phe/tyr (rs = 0.432, p < 0.05) but not with tyrosine concentrations. Neopterin concentrations were mean ± SEM 13.7 ± 3.31 nM (11/20 = 55% showed neopterin concentrations >8.6 nmol/L, the upper limit of the normal range). sTNF-R75 levels were 9.15 ± 0.70 ng/L (100% with sTNF-R75 concentrations >4.8 ng/L, the upper limit of the normal range). Neopterin concentrations were significantly higher in patients with higher FIGO stages (U = 2.46, p < 0.05; Table 2) and also correlated with FIGO stages (rs = 0.564, p < 0.01). sTNF-R75 concentrations did not differ between FIGO stages. There were no significant differences of marker concentrations in patients with different grading of the malignant process (Table 2), and also menopausal status did not influence the analytes studied (data not shown). Phenylalanine concentrations correlated with tyrosine concentrations (rs = 0.397) and with concentrations of sTNF-R75 (rs = 0.441; both p < 0.05) and with neopterin levels (rs = 0.346; p = 0.068; Table 3). Phe/tyr correlated even somewhat stronger than phenylalanine levels with
Table 2 Concentrations of phenylalanine and tyrosine, the phenylalanine to tyrosine ratio (Phe/tyr), 75 kD soluble tumor necrosis factor-a receptor (sTNF-R75), neopterin and isoprostane-8 concentrations in 20 patients with ovarian cancer compared according to their clinical presentation (mean ± SEM; *p < 0.05)
All FIGO: I–II, n = 6 III–IV, n = 14 Grade: 1 (n = 3) 2 (n = 8) 3 (n = 9)
Phenylalanine [lM]
Tyrosine [lM]
Phe/tyr [lM/lM]
sTNF-R75 [ng/L]
Neopterin [nM]
Isoprostane-8 [ng/L]
90.2 ± 4.38
62.2 ± 2.62
1.47 ± 0.07
9.15 ± 0.70
13.7 ± 3.31
139 ± 33.1
74.0 ± 6.95 96.0 ± 5.13*
58.1 ± 4.92 63.6 ± 3.60
1.29 ± 0.09 1.55 ± 0.10
7.83 ± 0.74 9.68 ± 0.93
6.61 ± 0.55 17.7 ± 5.01*
96.7 ± 38.7 141 ± 39.7
79.4 ± 17.2 91.0 ± 7.60 88.4 ± 6.70
62.5 ± 9.33 57.4 ± 3.64 67.1 ± 5.01
1.26 ± 0.09 1.61 ± 0.13 1.33 ± 0.08
7.43 ± 1.33 9.40 ± 1.16 9.21 ± 1.00
6.10 ± 1.15 19.1 ± 7.19 10.7 ± 2.12
36.0 ± 5.66 137 ± 40.6 146 ± 55.0
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Table 3 Correlation between concentrations of phenylalanine, tyrosine and the phenylalanine to tyrosine ratio (Phe/tyr), 75 kD soluble TNF-a receptor (sTNF-R75), neopterin and isoprostane-8 in 20 patients with ovarian cancer; Spearman rank correlation coefficients rs and p-values are shown (significant associations are printed in bold shape) Neopterin
Isoprostane-8
0.441 (0.03) 0.120 (n.s.) 0.549 (<0.01)
0.346 (0.07) 0.199 (n.s.) 0.497 (0.01)
0.450 (0.02) 0.146 (n.s.) 0.340 (0.07)
Isoprostane-8 [ng/L]
Phenylalanine Tyrosine Phe/tyr
sTNF-R75
1000
100
10 40
80
120
160
Phenylalanine[µM]
both, sTNF-R75 (rs = 0.549; p < 0.01) and neopterin (rs = 0.497; p = 0.01; Fig. 1). No such correlation with immune activation markers existed for tyrosine concentrations. Concentrations of sTNF-R75 and neopterin correlated strongly with each other (rs = 0.734, p < 0.001). Finally, 8-isoprostane concentrations in patients were 139 ± 148 ng/L (Table 1) and correlated with phenylalanine (rs = 0.450, p = 0.02; Fig. 2) but not with tyrosine concentrations. The correlation with phe/tyr did not quite reach statistical significance (rs = 0.340, p = 0.071). Isoprostane-8 was associated with sTNF-R75 (rs = 0.381, p < 0.05), but only tended to correlate with neopterin concentrations (rs = 0.312, p = 0.09).
Fig. 2. Association between serum phenylalanine and isoprostane-8 concentrations (rs = 0.450, p = 0.02; note log-scale for isoprostane-8 concentrations) in 20 patients with ovarian cancer (Note. log-scale for isoprostane-8 concentrations).
increased. Phe/tyr estimates PAH activity [10,16,18]; thus, the higher phe/tyr suggests an impaired conversion of phenylalanine to tyrosine by PAH. A reduced PAH activity could result from insufficient supply with cofactor BH4, which is required as a hydrogen donator for the hydroxylation of phenylalanine to tyrosine by PAH [9]. Indeed, BH4 is chemically very sensitive to oxidation and its oxidation is irreversible [14]. The association found between the impaired metabolism of phenylalanine with oxidative stress marker 8-isoprostane suggests that oxidative stress relates to BH4 deficiency, and BH4-dependent enzymes like PAH may miss their necessary cofactor [15]. In this case, tyrosine formation will decrease
4. Discussion
2.4
2.4
2.0
2.0
Phe/tyr[µM/µM]
Phe/tyr [µM /µ M]
Higher phenylalanine concentrations and higher phe/tyr were found in ovarian cancer patients with higher FIGO stage as compared with lower stage. In parallel to higher phenylalanine concentrations and higher phe/tyr, immune activation markers neopterin and sTNF-R75 were also found
1.6
1.2
0.8
1.6
1.2
0.8 4
6
8
10
12
sTNFR75 [ng/L]
14
16
18
1
10
100
Neopterin [nM]
Fig. 1. Association between serum phenylalanine to tyrosine ratio (Phe/tyr) and 75 kD soluble tumor necrosis factor-a receptor (sTNFR75; rs = 0.549, p < 0.01; left graph) and neopterin (rs = 0.497, p = 0.01; right graph) in 20 patients with ovarian cancer (Note. log-scale for neopterin concentrations).
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and phenylalanine concentrations are supposed to increase. But it has to be mentioned that tyrosine is not an end-product: tyrosine itself is substrate for a second enzymatic reaction, in which tyrosine hydroxylase, another BH4-dependent enzyme, forms L-DOPA [9]. So, one of the consequences of BH4 deficiency would be an impaired formation of the end products of this pathway, namely, adrenaline, noradrenaline, dopamine, and L-DOPA. The correlation found between phenylalanine concentrations, phe/tyr and sTNF-R75 and neopterin concentrations may provide an explanation for the disturbed PAH activity. sTNF-R75 is shed from TNF-receptor carrying cells upon activation [11]. Likewise, neopterin is released by monocytederived macrophages and dendritic cells upon proinflammatory stimuli like IFN-c [12]. Therefore, increased sTNF-R75 and neopterin concentrations, both indicate an activated pro-inflammatory immune response, which is supported by the rather close correlation found between the two compounds in our study. IFN-c is also a very potent stimulus for the production of reactive oxygen species by activated monocytic cells [13], and neopterin itself was found to enhance oxidizing capacity of ROS [20,21]. The chronic immune system activation appears to be a major cause of the loss of antioxidants and the development of oxidative stress [22]. Therefore, subnormal concentrations of antioxidants, like vitamin C and E, have been observed in diseases associated with immune activation and inflammation [23,24], and may also impair availability of BH4 in patients with malignancy [15]. Such conclusion is further supported by the correlation found between 8-isoprostane concentrations and concentrations of phenylalanine and sTNF-R75. 8Isoprostane is an indicator of the oxidative stress response, because it accumulates as a peroxidation product of arachidonic acid. When a higher degree of immune activation is associated with reduced conversion of phenylalanine to tyrosine one might expect that the association between immune activation markers and phenylalanine concentrations and phe/tyr might exist also in other pathological conditions such as infections, autoimmune syndromes or other types of cancer. In fact, earlier studies have described increased phenylalanine concentrations in patients suffering from infections, multiple trauma, sepsis and burns [1–6]. In the same clinical conditions also increased neopterin concentrations have been described [8,25–27]. Recently we observed an associ-
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ation between elevated neopterin concentrations and phenylalanine concentrations and phe/tyr in patients after multiple trauma [16]. One may speculate that a similar association between immune activation markers and increased phenylalanine concentrations can be detected in the other groups of patients as well. However, this still needs to be demonstrated. L-Phenylalanine is the precursor of tyrosine which is the precursor of DOPA and other neurotransmitters like dopamine, epinephrine and norepinephrine [9]. Earlier studies imply impaired metabolism of dopamine, and noradrenaline (norepinephrine) in mood disorders [9,19]. Clinical symptoms like depressive mood are more likely to develop in patients presenting with increased concentrations of phenylalanine, probably due to its impaired hydroxylation [19,28]. This fact might be of some relevance also in cancer patients. Depressive mood disorders probably develop in patients with increased phenylalanine concentrations, maybe due to an impaired hydroxylation of phenylalanine [9,19]. Patients with depression often show immune activation and increased neopterin concentrations [29,30], which may relate to disturbed BH4 and PAH activity [31]. It is known that BH4 can be stabilized with antioxidant vitamins like ascorbic acid, vitamin E (a-tocopherol) and carotinoides [32–34]. It would be worth testing if antioxidant supplementation is able to reduce phenylalanine in patients with moderate hyperphenylalaninemia. In conclusion, our study shows significant correlations between increased sTNF-R75 and neopterin concentrations, and phenylalanine, phe/tyr, and 8isoprostane in patients with ovarian cancer. Our results would be explainable by an impairment of PAH in these patients, and oxidative stress could be involved. Inflammation and immune activation may be involved in the disturbed phenylalanine conversion, and it needs to be shown whether this might relate to psychiatric abnormalities in patients. Notably, a correlation found does not necessarily relate to any cause-effect relationship and the number of patients investigated in this study is certainly too small for any final conclusion. Acknowledgements This work was supported by the ‘‘Stiftung Propter Homines, Vaduz -Fu¨rstentum Liechtenstein”. The authors thank Miss Astrid Haara for excellent technical assistance.
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Cancer Letters 272 (2008) 141–147 www.elsevier.com/locate/canlet
Serum phenylalanine concentrations in patients with ovarian carcinoma correlate with concentrations of immune activation markers and of isoprostane-8 Gabriele Neurauter a, Anna V. Grahmann b, Martin Klieber a, Alain Zeimet c, Maximilian Ledochowski d, Barbara Sperner-Unterweger b, Dietmar Fuchs a,* a
Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz Pregl Strasse 3, 6020 Innsbruck, Austria b Department of Psychiatry, Innsbruck Medical University, Innsbruck, Austria c Department of Gynaecology and Obstetrics, Innsbruck Medical University, Innsbruck, Austria d Department of Internal Medicine, Innsbruck Medical University, Innsbruck, Austria Received 27 May 2008; received in revised form 27 May 2008; accepted 3 July 2008
Abstract Increased blood concentrations of essential amino acid phenylalanine are common in patients with HIV infection, in trauma and sepsis and in patients with cancer. The reason for this phenomenon is still unclear. However, all these clinical conditions are known to be linked with inflammation and immune activation. Oxidative stress resulting from chronic immune activation and inflammation could impair activity of phenylalanine (4)-hydroxylase (PAH) and thus give rise to increased phenylalanine concentrations. We therefore examined in 20 patients with ovarian cancer a possible association of serum concentrations of phenylalanine and tyrosine with immune activation markers 75 kDa soluble tumor necrosis factor-a receptor (sTNF-R75) and neopterin, and of oxidative stress marker isoprostane-8. Phenylalanine concentrations were higher in patients with higher FIGO stage and correlated with concentrations of sTNF-R75 (rs = 0.441) and neopterin (rs = 0.346; both p < 0.05). No such correlations existed for tyrosine levels. The phenylalanine to tyrosine ratio (phe/tyr), an estimate of PAH activity, correlated somewhat stronger with sTNF-R75 (rs = 0.549; p < 0.01) and neopterin (rs = 0.497; p = 0.01). Finally, phenylalanine concentrations correlated with isoprostane-8 concentrations (rs = 0.450, p = 0.02). Correlations of phenylalanine and phe/tyr with immune activation markers point to a potential role of inflammation and immune activation in the accumulation of phenylalanine. The relationship between oxidative stress marker isoprostane-8 and phenylalanine as well as sTNF-R75 concentrations suggests a link between reactive oxygen species formed during chronic immune activation and inflammation and the decline of PAH activity, which might underlie the increase of phe/tyr (248 words). Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Phenylalanine; Tyrosine; Phenylalanine hydroxylase; Oxidative stress; Immune activation
*
Corresponding author. Tel.: +43 152 9003 70350; fax: +43 512 9003 73330. E-mail address: [email protected] (D. Fuchs).
0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.07.002
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1. Introduction Increased blood concentrations of essential amino acid phenylalanine have been described in consuming diseases such as HIV-1 infection, trauma, sepsis, burn, and malignancy [1–6]. The reason for this increase of phenylalanine is unexplained. However, these clinical conditions are known to be linked with inflammation and immune activation and with increased concentrations of immune activation markers such as serum soluble 75 kDa tumor necrosis factor-a (sTNF-R75) [7] and neopterin [8]. Subnormal enzymatic conversion of phenylalanine by phenylalanine-4-hydroxylase (PAH) might explain the accumulation of phenylalanine in patients [9,10]. Phenylalanine is the precursor of tyrosine, an important amino acid for the biosynthesis of neurotransmitters like L-DOPA (L3,4-dihydroxyphenylalanine) and catecholamines dopamine, epinephrine and norepinephrine. For enzymatic hydroxylation of phenylalanine to tyrosine by PAH, the cofactor 5,6,7,8-tetrahydrobiopterin (BH4), the reduced form of biopterin, is needed as a hydrogen donator [9]. TNF-a receptor (75 kDa) is shed from activated immunocompetent cells [7], and also neopterin is a product, which accumulates during states of immune activation, because large amounts of it are released by monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-c (IFN-c) [11,12]. Increased concentrations of sTNF-R75 and neopterin are common in the blood of patients suffering from conditions like virus infections, autoimmune syndromes and malignancy [7,8]. In parallel to neopterin and sTNF-R75 formation, macrophages and other cells stimulated with IFN-c produce reactive oxygen species (ROS) at a high rate [13]. Overwhelming production of ROS may wipe out antioxidant defence systems and, in turn, oxidative stress is developing. PAH cofactor BH4 is extremely sensitive to oxidation [14], and if BH4 is destroyed, the conversion rate of phenylalanine to tyrosine by PAH is reduced, it should be reflected by an increased phenylalanine to tyrosine ratio (phe/tyr), an estimate of PAH [10,15]. Oxidative stress due to chronic immune activation and inflammation could be involved in the increase of serum phenylalanine concentrations in patients. Recently we described a correlation between phe/tyr and neopterin concentrations in patients after multiple trauma [16].
To further substantiate the possible relationship between inflammation and immune activation and disturbed conversion of phenylalanine to tyrosine, we determined serum concentrations of these amino acids in 21 patients with ovarian carcinoma and compared the results with concentrations of inflammation and immune activation markers sTNF-R75 and neopterin and isoprostane-8, a peroxidation product of arachidonic acid, which represents a marker of oxidative stress [17]. 2. Materials and methods 2.1. Patients Twenty female patients (mean age: 53; range 27– 74 years) with ovarian cancer were recruited from the Department of Gynaecology at the University Clinics of Innsbruck, Austria. Five patients presented with FIGO stage Ib–Ic, 2 with FIGO IIa, 12 with FIGO IIIb–IIIc, and 1 with FIGO IV (Table 1). Degree of malignancy was Stage 1 in 3, Stage 2 in 8, and Stage 3 in 9. Twelve patients (61.9%) were postoperative without residual tumor, 8 (38.1%) were with residual tumor, 9 were premenopausal, 1 perimenopausal, and 10 postmenopausal. All participants’ rights were protected, and according to the Helsinki Declaration informed consent was obtained that a small portion of their blood collected for routine examinations was forwarded for further scientific testings. Table 1 Clinical data of 20 patients with ovarian cancer Number Tumor type
Stage
Malignancy grade
Post-operative status Menopausal status
Mucinous cystadenocarcinoma Serous cystadenocarcinoma Endometroid carcinoma Other FIGO Ib FIGO Ic FIGO IIa FIGO IIIb FIGO IIIc FIGO IV Grade 1 Grade 2 Grade 3 Residual tumor No residual tumor Premenopausal Perimenopausal Postmenopausal
3 15 1 1 3 2 1 1 12 1 3 8 9 12 8 9 1 10
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2.2. Laboratory examinations Blood serum samples were collected after surgical treatment of patients was performed and before chemotherapy was started. Samples were kept frozen at 20 °C until analysis. Phenylalanine and tyrosine concentrations were determined by high performance liquid chromatography monitoring their natural fluorescence at an excitation wavelength of 210 nm and an emission wavelength of 302 nm simultaneously [18]. Serum (100 ll) was diluted with 100 ll of 500 lM 3-nitro-L-tyrosine (internal standard) and 25 ll of 2 M trichloroacetic acid was used to precipitate and separate proteins. After centrifugation, supernatants of the samples were diluted 1:25 with 0.015 M potassium dihydrogen-phosphate, which was also used as elution buffer on HPLC. In parallel to the sera, an albumin-based calibration mixture was prepared which contained 100 lM phenylalanine and 100 lM tyrosine and underwent the same pre-analytical procedures as serum specimens. To estimate the activity of PAH, the ratio of the substrate phenylalanine versus the concentrations of the enzyme product tyrosine (=phe/tyr) was calculated [10,19]. Concentrations of sTNF-R75 (R&D Systems, Minneapolis, MN), neopterin (BRAHMS Diagnostica, Hennigsdorf/ Berlin, Germany) and isoprostane-8 (IBL, Hamburg, Germany) were measured by commercially available ELISAs. 2.3. Statistical analysis Concentrations of analytes were given as mean values ± SEM. Because some of the data did not show normal distribution, non-parametric Friedman test and Wilcoxon signed ranks test were applied for comparison of grouped data. Associa-
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tions between marker concentrations were calculated using Spearman rank correlation technique. The Statistical Package for the Social Sciences (version 14 SPSS, Chicago, Ill, USA) was used. p-values below 0.05 were considered to indicate significant differences and correlations. 3. Results Phenylalanine concentrations were 90.2 ± 4.38 lM and tyrosine concentrations were 62.2 ± 2.62 lM, phe/ tyr was 1.47 ± 0.071 (Table 1). Phenylalanine concentrations were significantly higher in patients with FIGO stage III and IV as compared to stages I and II (U = 2.154, p < 0.05; Table 2), whereas tyrosine concentrations did not differ between the two groups. Also phe/tyr was higher in FIGO stages III and IV than in patients with FIGO stage I and II, but the difference was only of borderline significance (U = 1,78, p = 0.075). There were significant correlations between FIGO stage and phenylalanine concentrations (rs = 0.523, p = 0.01) and phe/tyr (rs = 0.432, p < 0.05) but not with tyrosine concentrations. Neopterin concentrations were mean ± SEM 13.7 ± 3.31 nM (11/20 = 55% showed neopterin concentrations >8.6 nmol/L, the upper limit of the normal range). sTNF-R75 levels were 9.15 ± 0.70 ng/L (100% with sTNF-R75 concentrations >4.8 ng/L, the upper limit of the normal range). Neopterin concentrations were significantly higher in patients with higher FIGO stages (U = 2.46, p < 0.05; Table 2) and also correlated with FIGO stages (rs = 0.564, p < 0.01). sTNF-R75 concentrations did not differ between FIGO stages. There were no significant differences of marker concentrations in patients with different grading of the malignant process (Table 2), and also menopausal status did not influence the analytes studied (data not shown). Phenylalanine concentrations correlated with tyrosine concentrations (rs = 0.397) and with concentrations of sTNF-R75 (rs = 0.441; both p < 0.05) and with neopterin levels (rs = 0.346; p = 0.068; Table 3). Phe/tyr correlated even somewhat stronger than phenylalanine levels with
Table 2 Concentrations of phenylalanine and tyrosine, the phenylalanine to tyrosine ratio (Phe/tyr), 75 kD soluble tumor necrosis factor-a receptor (sTNF-R75), neopterin and isoprostane-8 concentrations in 20 patients with ovarian cancer compared according to their clinical presentation (mean ± SEM; *p < 0.05)
All FIGO: I–II, n = 6 III–IV, n = 14 Grade: 1 (n = 3) 2 (n = 8) 3 (n = 9)
Phenylalanine [lM]
Tyrosine [lM]
Phe/tyr [lM/lM]
sTNF-R75 [ng/L]
Neopterin [nM]
Isoprostane-8 [ng/L]
90.2 ± 4.38
62.2 ± 2.62
1.47 ± 0.07
9.15 ± 0.70
13.7 ± 3.31
139 ± 33.1
74.0 ± 6.95 96.0 ± 5.13*
58.1 ± 4.92 63.6 ± 3.60
1.29 ± 0.09 1.55 ± 0.10
7.83 ± 0.74 9.68 ± 0.93
6.61 ± 0.55 17.7 ± 5.01*
96.7 ± 38.7 141 ± 39.7
79.4 ± 17.2 91.0 ± 7.60 88.4 ± 6.70
62.5 ± 9.33 57.4 ± 3.64 67.1 ± 5.01
1.26 ± 0.09 1.61 ± 0.13 1.33 ± 0.08
7.43 ± 1.33 9.40 ± 1.16 9.21 ± 1.00
6.10 ± 1.15 19.1 ± 7.19 10.7 ± 2.12
36.0 ± 5.66 137 ± 40.6 146 ± 55.0
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Table 3 Correlation between concentrations of phenylalanine, tyrosine and the phenylalanine to tyrosine ratio (Phe/tyr), 75 kD soluble TNF-a receptor (sTNF-R75), neopterin and isoprostane-8 in 20 patients with ovarian cancer; Spearman rank correlation coefficients rs and p-values are shown (significant associations are printed in bold shape) Neopterin
Isoprostane-8
0.441 (0.03) 0.120 (n.s.) 0.549 (<0.01)
0.346 (0.07) 0.199 (n.s.) 0.497 (0.01)
0.450 (0.02) 0.146 (n.s.) 0.340 (0.07)
Isoprostane-8 [ng/L]
Phenylalanine Tyrosine Phe/tyr
sTNF-R75
1000
100
10 40
80
120
160
Phenylalanine[µM]
both, sTNF-R75 (rs = 0.549; p < 0.01) and neopterin (rs = 0.497; p = 0.01; Fig. 1). No such correlation with immune activation markers existed for tyrosine concentrations. Concentrations of sTNF-R75 and neopterin correlated strongly with each other (rs = 0.734, p < 0.001). Finally, 8-isoprostane concentrations in patients were 139 ± 148 ng/L (Table 1) and correlated with phenylalanine (rs = 0.450, p = 0.02; Fig. 2) but not with tyrosine concentrations. The correlation with phe/tyr did not quite reach statistical significance (rs = 0.340, p = 0.071). Isoprostane-8 was associated with sTNF-R75 (rs = 0.381, p < 0.05), but only tended to correlate with neopterin concentrations (rs = 0.312, p = 0.09).
Fig. 2. Association between serum phenylalanine and isoprostane-8 concentrations (rs = 0.450, p = 0.02; note log-scale for isoprostane-8 concentrations) in 20 patients with ovarian cancer (Note. log-scale for isoprostane-8 concentrations).
increased. Phe/tyr estimates PAH activity [10,16,18]; thus, the higher phe/tyr suggests an impaired conversion of phenylalanine to tyrosine by PAH. A reduced PAH activity could result from insufficient supply with cofactor BH4, which is required as a hydrogen donator for the hydroxylation of phenylalanine to tyrosine by PAH [9]. Indeed, BH4 is chemically very sensitive to oxidation and its oxidation is irreversible [14]. The association found between the impaired metabolism of phenylalanine with oxidative stress marker 8-isoprostane suggests that oxidative stress relates to BH4 deficiency, and BH4-dependent enzymes like PAH may miss their necessary cofactor [15]. In this case, tyrosine formation will decrease
4. Discussion
2.4
2.4
2.0
2.0
Phe/tyr[µM/µM]
Phe/tyr [µM /µ M]
Higher phenylalanine concentrations and higher phe/tyr were found in ovarian cancer patients with higher FIGO stage as compared with lower stage. In parallel to higher phenylalanine concentrations and higher phe/tyr, immune activation markers neopterin and sTNF-R75 were also found
1.6
1.2
0.8
1.6
1.2
0.8 4
6
8
10
12
sTNFR75 [ng/L]
14
16
18
1
10
100
Neopterin [nM]
Fig. 1. Association between serum phenylalanine to tyrosine ratio (Phe/tyr) and 75 kD soluble tumor necrosis factor-a receptor (sTNFR75; rs = 0.549, p < 0.01; left graph) and neopterin (rs = 0.497, p = 0.01; right graph) in 20 patients with ovarian cancer (Note. log-scale for neopterin concentrations).
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and phenylalanine concentrations are supposed to increase. But it has to be mentioned that tyrosine is not an end-product: tyrosine itself is substrate for a second enzymatic reaction, in which tyrosine hydroxylase, another BH4-dependent enzyme, forms L-DOPA [9]. So, one of the consequences of BH4 deficiency would be an impaired formation of the end products of this pathway, namely, adrenaline, noradrenaline, dopamine, and L-DOPA. The correlation found between phenylalanine concentrations, phe/tyr and sTNF-R75 and neopterin concentrations may provide an explanation for the disturbed PAH activity. sTNF-R75 is shed from TNF-receptor carrying cells upon activation [11]. Likewise, neopterin is released by monocytederived macrophages and dendritic cells upon proinflammatory stimuli like IFN-c [12]. Therefore, increased sTNF-R75 and neopterin concentrations, both indicate an activated pro-inflammatory immune response, which is supported by the rather close correlation found between the two compounds in our study. IFN-c is also a very potent stimulus for the production of reactive oxygen species by activated monocytic cells [13], and neopterin itself was found to enhance oxidizing capacity of ROS [20,21]. The chronic immune system activation appears to be a major cause of the loss of antioxidants and the development of oxidative stress [22]. Therefore, subnormal concentrations of antioxidants, like vitamin C and E, have been observed in diseases associated with immune activation and inflammation [23,24], and may also impair availability of BH4 in patients with malignancy [15]. Such conclusion is further supported by the correlation found between 8-isoprostane concentrations and concentrations of phenylalanine and sTNF-R75. 8Isoprostane is an indicator of the oxidative stress response, because it accumulates as a peroxidation product of arachidonic acid. When a higher degree of immune activation is associated with reduced conversion of phenylalanine to tyrosine one might expect that the association between immune activation markers and phenylalanine concentrations and phe/tyr might exist also in other pathological conditions such as infections, autoimmune syndromes or other types of cancer. In fact, earlier studies have described increased phenylalanine concentrations in patients suffering from infections, multiple trauma, sepsis and burns [1–6]. In the same clinical conditions also increased neopterin concentrations have been described [8,25–27]. Recently we observed an associ-
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ation between elevated neopterin concentrations and phenylalanine concentrations and phe/tyr in patients after multiple trauma [16]. One may speculate that a similar association between immune activation markers and increased phenylalanine concentrations can be detected in the other groups of patients as well. However, this still needs to be demonstrated. L-Phenylalanine is the precursor of tyrosine which is the precursor of DOPA and other neurotransmitters like dopamine, epinephrine and norepinephrine [9]. Earlier studies imply impaired metabolism of dopamine, and noradrenaline (norepinephrine) in mood disorders [9,19]. Clinical symptoms like depressive mood are more likely to develop in patients presenting with increased concentrations of phenylalanine, probably due to its impaired hydroxylation [19,28]. This fact might be of some relevance also in cancer patients. Depressive mood disorders probably develop in patients with increased phenylalanine concentrations, maybe due to an impaired hydroxylation of phenylalanine [9,19]. Patients with depression often show immune activation and increased neopterin concentrations [29,30], which may relate to disturbed BH4 and PAH activity [31]. It is known that BH4 can be stabilized with antioxidant vitamins like ascorbic acid, vitamin E (a-tocopherol) and carotinoides [32–34]. It would be worth testing if antioxidant supplementation is able to reduce phenylalanine in patients with moderate hyperphenylalaninemia. In conclusion, our study shows significant correlations between increased sTNF-R75 and neopterin concentrations, and phenylalanine, phe/tyr, and 8isoprostane in patients with ovarian cancer. Our results would be explainable by an impairment of PAH in these patients, and oxidative stress could be involved. Inflammation and immune activation may be involved in the disturbed phenylalanine conversion, and it needs to be shown whether this might relate to psychiatric abnormalities in patients. Notably, a correlation found does not necessarily relate to any cause-effect relationship and the number of patients investigated in this study is certainly too small for any final conclusion. Acknowledgements This work was supported by the ‘‘Stiftung Propter Homines, Vaduz -Fu¨rstentum Liechtenstein”. The authors thank Miss Astrid Haara for excellent technical assistance.
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