Decreased phenylalanine uptake and turnover in patients with vitiligo

Molecular Genetics and Metabolism 86 (2005) S27–S33 www.elsevier.com/locate/ymgme

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Decreased phenylalanine uptake and turnover in patients with vitiligo Karin U. Schallreuter a,b,¤, Bhaven Chavan a, Hartmut Rokos b, Nigel Hibberts c, Angela Panske b, John M. Wood a,c b

a Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, UK Institute for Pigmentary Disorders in association with EM Arndt University Greifswald, Germany and University of Bradford, UK c Biomedical Sciences, University of Bradford, UK

Received 3 May 2005; received in revised form 1 July 2005; accepted 6 July 2005 Available online 6 September 2005

Abstract The human epidermis has the full machinery for autocrine L-phenylalanine turnover to L-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I–VI, Fitzpatrick classiWcation) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) signiWcantly increases epidermal PAH activities 24 h after exposure. Importantly, L-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired L-phenylalanine turnover in the skin as well as in serum because aVected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with L-phenylalanine (100 mg/kg body weight), 29.6% of 970 patients tested (n D 287/970) yielded serum phenylalanine/tyrosine ratios 74 and 35.3% (n D 342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n D 90/970) had both serum L-phenylalanine levels 72.0 mg/dl and phe/tyr ratios 74.0. Isolated HPA was found in 26% (n D 252/970), whereas 20.3% had only increased ratios (n D 197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro L-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a signiWcantly decreased calcium dependent L-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could oVer an additional novel mechanism in the aetiology of HPA.  2005 Elsevier Inc. All rights reserved. Keywords: 6-Tetrahydrobiopterin; Hyperphenylalaninaemia; Phenylalanine hydroxylase; L-Phenylalanine; L-Tyrosine; Phenylalanine loading test; Melanogenesis; H2O2; Vitiligo; Calcium uptake

Introduction The human skin holds the capacity for (6R)-Lerythro-5,6,7,8-tetrahydrobiopterin (6BH4) de novo synthesis/recycling/regulation in keratinocytes and in the pigment forming melanocyte [1]. Cofactor levels as well as the rate limiting step for epidermal 6BH4 synthesis via *

Corresponding author. Fax: +44 1274 236489. E-mail address: [email protected] (K.U. Schallreuter). 1096-7192/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.07.023

GTP-cyclohydrolase I (EC 3.5.4.16,GTP-CH I), followed by phenylalanine hydroxylase (EC 1.14.16.1, PAH) and 4a-carbinolamine dehydratase (EC 4.2.1.96, PCD) activities, correlate with diVerent inherited skin colour (skin phototypes I–VI, Fitzpatrick classiWcation) [2,3]. Moreover, it was demonstrated that exposure to UVB–light increases GTP-CH I activities in association with signiWcantly higher 6BH4 levels after 24 h [2]. This was also the case for PAH and PCD [2]. From those results it was apparent that the entire 6BH4 system was involved in the pigmentation process.

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Human melanocytes are derived from the neural crest and these cells are responsible for biosynthesis and distribution of the melanins in the skin, hair, eye and ear [4]. However, besides a complicated pathway, the initiation of pigmentation relies on both suYcient supply of L-tyrosine and the expression/activity of the key enzymes tyrosinase (EC 1.14.18.1) and tyrosine hydroxylase isoform I (EC 1.14.16.2, TH) in these special cells [5,6]. The process of melanogenesis occurs within speciWc organelles, the melanosomes. In this context it is important that epidermal melanocytes reside in an avascular compartment. Hence, these cells need other mechanisms in place to drive cellular metabolic events. In order to promote melanogenesis, two amino acids are of major importance i.e. L-phenylalanine and Ltyrosine. The latter is the substrate for both tyrosinase and tyrosine hydroxylase isoform I (TH) and its uptake occurs by facilitated diVusion [7,8]. Concentrations in the blood stream range around 50 £ 10¡6 M and those are suYcient to drive TH activities but they are too low for tyrosinase to initiate melanogenesis [9]. In this context it is of interest that L-phenylalanine concentrations in the serum vary from 70–90 £ 10¡6 M [10,11]. Moreover, melanocytes have very high cytosolic PAH activities even comparable to those in hepatocytes [1]. In fact it has been shown under in vitro conditions that 65% of the melanin formed originates from L-phenylalanine as demonstrated by 14C-labelled L-phenylalanine and 3Hlabelled L-tyrosine uptake into melanocytes [11]. Fig. 1 L-PHENYLALANINE O2

PHENYLALANINE HYDROXYLASE

6BH4 L-TYROSINE O2

TYROSINE HYDROXYLASE

6BH4

Phenylalanine Hydroxylase Activity [nmol/mg protein/min]

S28

30

26.4 21.8 17.9

20 20 12.6 7.8

10 10 4.2 00

I

II

III

IV

V

VI

Skin phototype Fig. 2. Epidermal PAH activities correlate with skin colour. Epidermal phenylalanine hydroxylase activities (nmol/mg protein/min) in skin phototype I–VI, Fitzpatrick classiWcation increase linearly from fair to dark skin. Enzyme activities were determined in crude cell extracts from epidermal suction blister tissue obtained by the method of Kiistala [42]. Enzyme assays were performed following the conversion of 14 C L-phenylalanine to 14C L-tyrosine using thin layer chromatography as described in detail [1,2].

presents the modiWed Raper–Mason scheme for melanogenesis with special emphasis towards the important step of intracellular L-phenylalanine turnover via PAH in the cytosol followed by the formation of L-dopa by TH isoform I in the melanosome, both controlled by 6BH4 [5]. In addition this cofactor is an allosteric inhibitor of tyrosinase in 10¡6 M range [12]. PAH activities increase linearly with inherited skin colour (skin phototypes I–VI, Fitzpatrick classiWcation) (Fig. 2) [1,2]. Moreover, increased epidermal turnover of phenylalanine after UVB exposure with one individual MED was observed in vitro in epidermal suction blister tissue and by in vivo FT-Raman spectroscopy (Fig. 3) [2,13]. Taken together, the uptake/transport and turnover of the neutral amino acid L-phenylalanine are crucial steps for both formation of inherited skin colour and de novo melanin synthesis (i.e. tanning process).

L-DOPA O2 6BH4

TYROSINASE

DOPAQUINONE

CYSTEINYLDOPA

Vitiligo – a model for perturbed L-phenylalanine uptake and metabolism? EUMELANIN (BLACK)

PHEOMELANIN (RED)

Fig. 1. The modiWed Raper–Mason scheme of melanogenesis. L-Phenylalanine is converted to L-tyrosine in the presence of 6BH4 and oxygen in the cytosol of melanocytes followed by L-dopa formation via tyrosine hydroxylase isoform I in the acidic environment of the melanosome again in the presence of 6BH4 and oxygen. After activation of tyrosinase by L-dopa, dopaquinone is formed yielding Wnally the end products pheo- and eumelanin. Tyrosinase can be allosterically controlled by 6BH4. N.B. This scheme highlights the importance of PAH for the L-tyrosine supply in melanocytes.

In the depigmentation disorder vitiligo the epidermal as well as the systemic uptake and turnover of L -phenylalanine is altered [1,14]. The reason is yet not fully understood. Vitiligo is a disease with an acquired sudden loss of skin colour aVecting 0.5–1% of all races. Besides the loss of pigment, the majority of these patients are otherwise healthy. There is no gender predominance. The age of onset can vary from few days post-partum to old age. The family history is positive in 35–40% of aVected individuals. The cause of the disease is still unknown

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16

[nmol/mg protein/min]

Phenylalanine Hydroxylase Activity

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n = 10

n = 10

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0 0

Time after UVB [Hours]

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Phe 1004 cm-1

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0 1140

Tyr 846 cm-1

1040

940 Wavelength

840

740

[cm-1]

Fig. 3. In vitro and in vivo evidence for increased phenylalanine hydroxylase activity in the human epidermis after UVB exposure: (A) signiWcant increase of epidermal PAH activities 24 h after UVB exposure (311 nm, 1 MED) in skin phototype III (Fitzpatrick classiWcation). The result is based on analysis of 10 healthy volunteers as described earlier [1,2] and (B) in vivo epidermal phenylalanine turnover by FT-Raman spectroscopy after UVB (1 MED). Note the decrease of phenylalanine at 1004 cm¡1 (—) in association with an increase of tyrosine at 846 cm¡1 (- - -).

[15]. There are several hypotheses trying to explain this enigma but none of them can satisfy the plethora of diVerent Wndings. The most favoured theory is still the autoimmune hypothesis, followed by various other theories including oxidative stress by hydrogen peroxide (H2O2), catecholamine and cholinergic imbalance, toxic metabolites of the melanogenic pathway, virus and hormones [16]. More recent research has focussed on genetic aspects [17,18].

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individuals were recognised already several decades ago [23–25]. However, until recently the cause of these observations remained unknown [26]. It was shown that these cellular changes were associated with epidermal H2O2 accumulation [26]. Moreover, epidermal H2O2 concentrations in the 10¡3 M range were demonstrated by in vivo FT-Raman spectroscopy in acute/progressive vitiligo in all patients tested (n D 110)[19]. This ROS was assigned based on its O–O stretch at 875 cm¡1 [19]. The epidermal redox imbalance in these patients leads to decreased catalase, glutathione peroxidase and thioredoxin reductase levels which in turn amounts to even higher H2O2 concentrations [27–29]. Moreover, accumulation of oxidised pterins yields a characteristic Xuorescence under Wood’s light (351 nm) examination, which is absent in depigmented skin of other origin (Fig. 4) [1]. These oxidised pterins were identiWed as 6biopterin, 7-biopterin and pterin-6-carboxylic acid [1,20]. In this context it has been demonstrated that the entire epidermal 6BH4 de novo synthesis and recycling is aVected in vitiligo [1]. Only recently it was recognised that both 6BH4 recycling enzymes i.e. PCD and dihydropteridine reductase (EC 1.6.99.7, DHPR) are deactivated in their active sites due to oxidation of tryptophan and methionine residues by H2O2 concentrations in the 10¡3 M range [30,31]. Detailed enzyme kinetics with pure recombinant enzymes revealed upregulation with H2O2 concentrations in the 10¡6 M range [30,31]. Due to almost absent PCD activity in the epidermal compartment of these patients the appearance of 7biopterin was not surprising [1,32,33]. Moreover, PCD is tightly coupled to PAH, increasing its activity sevenfold. Since 7BH4 can inhibit PAH, it was also not surprising to Wnd low epidermal PAH activities in these patients (Fig. 5) [1,32,33]. Therefore, accumulation of A

B

Evidence for oxidative stress by H2O2 in vitiligo The loss of existing skin colour from the epidermis suggested the involvement of reactive oxygen species (ROS) [19,20]. Nowadays there is no doubt that these patients are subject to continuous oxidative stress by H2O2 (for review see [16]) [19–22]. In fact cellular vacuolation and granular deposits in epidermal cells of those

Fig. 4. Characteristic Xuorescence of vitiligo due to oxidised pterins: (A) vitiligo of the leg and (B) the same case under Wood’s (351 nm) light examination.

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600 500 400

287 bp

300 200 100

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kDa 200 140 100 80 60 50 40

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Phenylalanine Hydroxylase Activity [nmol/mg protein/min]

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The inXuence of calcium on cellular phenylalanine – uptake Previously it has been reported that L-phenylalanine uptake depends on the concentration of extra-cellular calcium as observed in primary epidermal cell cultures of keratinocytes and melanocytes [11,34,35]. In this context it was shown that these epidermal cells established from skin of patients with vitiligo revealed a signiWcantly decreased 45calcium uptake compared to healthy controls [36,37]. A comparative study on the inXuence of calcium on 3H-labelled L-phenylalanine uptake and its intracellular turnover to the labelled product L-tyrosine in primary epidermal melanocytes from patients and controls conWrmed the slow uptake of this important second messenger in these cells as described earlier [37]. In addition, we here demonstrate for the Wrst time that both 3H-labelled L-phenylalanine uptake and its turnover to 3H-labelled L-tyrosine is profoundly aVected by calcium in vitiligo melanocytes compared to controls (Fig. 6). Since L-phenylalanine is actively transported by LATS (large neutral amino acid transporter) coupled to the Na+/Ca2+ ATPase-antiporter, it remains to be shown, whether the breakdown in calcium homeostasis is due to altered calcium channels or to the ATPase eZux or to both [11,38].

Evidence for slow L-phenylalanine clearance in serum of patients with vitiligo

6

4

2

0 CONTROL

lesional non-lesional VITILIGO

Fig. 5. The presence of phenylalanine hydroxylase in the human epidermis: (A) mRNA expression of PAH by RT-PCR: lane 1, vitiligo melanocyte; lane 2, normal melanocyte; lane 3, vitiligo keratinocyte; lane 4, normal keratinocyte; lane 4, bp ladder; (B) Western blot analysis conWrms the presence of PAH in epidermal melanocytes and keratinocytes: lane 1, MW ladder; lane 2, melanocyte; lane 3, undiVerentiated keratinocyte; lane 4, diVerentiated keratinocyte and (C) decreased epidermal enzyme activities in patients with vitiligo (n D 8) compared to healthy controls (n D 10) skin phototype III (Fitzpatrick classiWcation).

L-phenylalanine

was expected and this assumption was indeed conWrmed by in vivo FT-Raman spectroscopy following the phenylalanine ring breathing mode at 1004 cm¡1 [14].

Recently it was recognised that vascular DHPR activities in patients with vitiligo are signiWcantly aVected by transfer of epidermal H2O2 [31]. Therefore it was tempting to examine PAH activities via the systemic L-phenylalanine uptake/clearance in these patients. In order to address this issue, we employed the oral loading test with L-phenylalanine (100 mg/kg body weight). Both L-tyrosine and L-phenylalanine concentrations were determined in serum after overnight fasting before and after loading with L-phenylalanine at 0, 1, 2 and 4 h by amino acid analysis (LKB 4151 alpha plus, Pharmacia LKB) as described earlier [13,39]. A quotient of L-phenylalanine over L-tyrosine (phe/tyr ratio) 64.0 was indicative for normal PAH activity [39]. Since a quotient between 4.0– 4.2 could present an overlapping area between normal and elevated ratios, these values were taken into consideration in the Wnal analysis [39]. Basic L-phenylalanine levels were regarded as normal 62.0 mg/dl (120
mol/l) [40]. This study included 970 patients (569 females, 401 males) with vitiligo and 53 matched adult controls. Patients were grouped into children (612 years, n D 43), adolescents (13–21years, n D 86) and adults (722 years, n D 841). The mean age was 37.5 years (range 4–71). The results showed that 287 from 970 patients had a phe/tyr

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uptake µmol/mg protein/10 min

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phe/tyr ≥4

10 1

45Ca

20

20 0

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30

0

30

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[3H]L-Phe uptake µmol/mg protein/10 min

90

B

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40 30 20 10 0

children (n=43)

8 7

adolescents (n=86)

adults (n=841)

Fig. 7. Age dependent distribution of slow L-phenylalanine turnover (phe/tyr ratio 74.0) and hyperphenylalaninaemia (serum phenylalanine 72.0 mg/dl) in patients with vitiligo (n D 970): (A) distribution of isolated HPA in children, adolescents and adults; (B) distribution of ratios 74.0 in children, adolescents and adults and (C) distribution of patients with both HPA and phe/tyr ratio 74.0.

6 5 4 3 2 1 0 1

1.5

10-3 M

2

45Calcium

Fig. 6. The inXuence of 45calcium uptake on [3H]L-phenylalanine and its turnover to [3H]L-tyrosine in melanocytes from controls (䊏) and patients with vitiligo (䊐) in the presence of increasing extra-cellular calcium concentrations: (A) [3H]L-phe uptake; (B) [3H]L-tyr formation and (C) 45Ca uptake From these experiments it can be concluded that the cellular L-phenylalanine uptake, its turnover to L-tyrosine and the calcium uptake are severely aVected in melanocytes of patients compared to controls.

ratio 74.0 (range 4.0–9.8). The distribution of the increased phe/tyr ratio in diVerent age groups is presented in Fig. 7A. Analysis of basic L-phenylalanine levels in serum after fasting overnight showed that 35.3% (n D 342/970) of all patients had mild to moderate hyperphenylalaninaemia (HPA) with levels ranging from 2 to 12.1 mg/dl (Fig. 7B).

In order to test, whether elevated serum phenylalanine levels corresponded to high phe/tyr ratios, both results were analysed in all three age groups. None of the children with elevated basic phenylalanine levels had a high phe/tyr ratio. In the adolescent group, 5 from 25 had both elevated phenylalanine levels and a high ratio, while 14 had only a phe/tyr ratio 74.0. Analysis of the adult group showed that 85 (85/297) patients had both, phenylalanine levels 72.0 mg/dl together with a phe/tyr ratio 74.0. 9.7% (n D 94/970) had phe/tyr ratios in the overlapping area (range 4.0– 4.2), leaving 19.9% (n D 193/970) with a signiWcant elevated ratio (range 4.2–9.8). Importantly, none of the patients had a history of phenylketonuria nor was this disorder running in their families. Moreover, there was also no sign for an impaired IQ in these patients [41]. To sum up, 20% of all patients with vitiligo demonstrated a signiWcant altered phe/tyr turnover and 35% showed a mild/moderate HPA without any clinical symptoms.

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Considering the above results, a mutation in the PAH gene seemed to be a reasonable explanation. To test this assumption analysis of the entire PAH gene took place in three diVerent laboratories. Surprisingly the results did not reveal any evidence for a genetic defect (Spritz RA et al., unpublished results; McCormack WT, personal communication, Guttler H and Schallreuter KU, unpublished results). In this context it was also suggested that the PCD gene could be aVected because untreated patients presented extremely low or completely absent epidermal enzyme activities in association with high 7BH4 levels [1]. However, analysis of the PCD gene in 10 patients with vitiligo showed only wild-type enzyme [30]. Later it was recognised that the low epidermal enzyme activities were due to H2O2 accumulation in the 10¡3 M range yielding deactivation of the enzyme active site [30]. Enzyme activities were restored after removal of this ROS with a topical applied pseudocatalase PC-KUS whereas low PAH activities were not aVected in this compartment. Since systemic DHPR activities recover after reduction/removal of epidermal H2O2 by this topical treatment modality [31], it was tempting to re-test serum phe/ tyr ratios of treated patients (n D 32) who had an initial ratio 74.0 before treatment. This study showed no diVerence in the ratios between before and after treatment in these patients. Based on these results we can conclude that neither systemic nor epidermal PAH activities unlike DHPR are directly aVected by reduction/removal of epidermal H2O2 by pseudocatalase PC-KUS in patients with vitiligo [16]. In summary, the results of this study on 970 patients demonstrated a mild to moderate HPA in approx. 35% of this cohort. Moreover, with increasing age the ability of L-phenylalanine turnover slows down considerably in this patient group. Furthermore, the in vitro data on epidermal melanocytes and keratinocytes established from aVected individuals also indicated that L-phenylalanine uptake/transport may play a major role due to impaired calcium homeostasis in this disorder [11,36,37]. Based on the phenylalanine loading test we identiWed three diVerent subgroups. One subgroup of patients with vitiligo showed both mild/moderate HPA together with a slow phenylalanine turnover despite normal serum 6BH4 levels. A second subgroup presents only a slow phenylalanine turnover with ratios >4.2, while a third subgroup may include a borderline group with overlapping ratios between 4.0 and 4.2. Whether the impaired L-phenylalanine uptake due to a perturbed cellular calcium homeostasis as shown under in vitro conditions can explain the isolated HPA in patients with vitiligo needs further investigation. Clearly L-phenylalanine uptake is facilitated by calcium and a perturbed calcium uptake/eZux is associated with a slow L-phenylalanine uptake as shown in this report.

The prolonged clearance of L-phenylalanine in the serum of all patients after oral loading and the persistent high levels in the epidermal compartment, as demonstrated by in vivo FT-Raman spectroscopy, support a slow uptake/clearance for this amino acid in this disease which could be fostered by impaired calcium uptake/ eZux. It is tempting to speculate that the above mechanism could add a novel mechanism in the understanding for isolated HPA. Future work is needed to understand this puzzle in more detail.

Acknowledgment This research was supported by a grant from Stiefel International to KUS. The PAH antibody was a kind gift from Professor A. Martinez, University of Bergen, Norway.

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