This hypothesis has been tested very recently using noninvasive Fourier Transform Raman spectroscopy directly in vivo on patients ( n = 23) and on age and sex matched control skin ( n = 10). The results demonstrated unam- biguously that all patients had higher levels of phenylalanine in their white skin compared to their normal skin (Fig. 19.4a). This observation was further substantiated with in vivo studies, where the individual phenylala- nine hydroxylase activities were followed in patients ( n = 32) and in con- trols ( n = 10) over 4 hours after a single oral loading with L-phenylalanine.
The results showed that all patients had a slower turnover from L-
phenylalanine to L-tyrosine, but 41 % of the patients were significantly slower (P = 0.0001). However, all patients returned to normal levels after 4 hours, underlining that these patients do not express a peripheral hyper- phenylalaninemia under normal conditions (Schallreuter et al. 1998) (Fig.
19.4b). These results also confirmed earlier studies, where patients with vitiligo never displayed any evidence for a peripheral accumulation of this essential amino acid (Cormane et al. 1985).
CHAPTER 19
Biochemical The0 y
155
CHAPTER 19
Biochemical Theory
Control skin
Normal Vitiligo
Patient skin
Fig. 19.4 (a) Epidermal phenylalanine levels. IT-Raman spectroscopy was utilized in vivo to determine epidermal phenylalanine concentrations. All patients (n = 23) showed higher levels in their involved skin compared to their uninvolved skin. Levels between healthy controls and uninvolved skin did not differ significantly. (b) L-phenylalanine turnover in vitiligo patients and controls. After a single oral loading with L-phenylalanine (100 mg/kg), the turnover of this essential amino acid to L-tyrosine was followed over four hours in 32 patients and 10 healthy controls. In all vitiligo patients, L-phenylalanine clearance was significantly slower than in controls, returning almost back to normal after four hours. The patient group could be divided into two subsets with 13 patients yielding phenylalanine to tyrosine ratios between 4.0 and 7.0
(en,
and the remaining 19 patients revealing a normal phenylalanine to tyrosine ratio, <4.0. This second subset had a less hindered L-phenylalanine clearance (A-A) compared to controls (0-0).18
16
14
12
- E
0
10
I
i?
0 C m
.-
- < 8
C
c 0
SL 6
4
2
0
(b)
CHAPTER 1 9
Biochemical Theory
T
I I I I
60 120 180 240
l i m e (minutes)
Fig. 19.4 Continued.
One consequence of increased epidermal L-phenylalanine levels would be the positive feedback on the de novo synthesis of 6BH4, via the regulatory protein GFRP (Harada et al. 1993). This would lead to increased activity of GTP-cyclohydrolase I, and therefore enhance the production of 6BH4. This cascade of events has been observed in vitiligo. Under physiological condi- tions, once there is sufficient amount of cofactor, enzyme activities should be down-regulated via the regulatory protein GFRP. However, this step does not appear to function properly in vitiligo because concentrations of 6BH4 accumulate to approximately 10-6 M. In addition the defective recy- cling of 6BH4 leads to the formation of micromolar levels of 7BH4 and H,O, in the epidermis of these patients (Schallreuter et al. 1994b, c). The oxidative stress in vitiligo caused by H,O, will be discussed in more detail in our
CHAPTER 19
BioChemicd Theory
chapter on pseudocatalase (Chapter 23). However, there is increasing evi- dence that epidermal 7BH,-inhibited phenylalanine hydroxylase consider- ably hampers the epidermal homeostasis for L-phenylalanine conversion to L-tyrosine in vitiligo (see Fig. 19.3).
One intriguing question remains. Are there still melanocytes present in the white skin of patients with vitiligo? Le Poole et al. (1993b) re-examined this issue using extensive histochemical and immunohistochemical methodology. Based on their results, this group concluded that there is no evidence for melanocytes in the white skin of these patients. Our own results contradict this conclusion. Recently we have found that decreased numbers of melanocytes are still present in the white skin, and that these cells can produce pigment under in vivo and in vitro conditions after correc- tion of the impaired homeostasis in patients skin or under culture condi- tions (Tobin & Schallreuter 1997). Our examination was conducted on 14 randomly selected patients with vitiligo, where the duration of the disease ranged from three to 20 years.
Summary
In summary, the above observations shed some new light on the aetiology of this disease. At the present time at least four possible consequences arise from altered tetrahydrobiopterin homeostasis in vitiligo:
1 the GFRP/6BH4binding is defective,
2 7BH, competes with 6BH4 for binding on GFRP,
3 7BH4 inhibited phenylalanine hydroxylase is the target, or
4 inefficient recycling of 6BH, due to very low 4a-OH-tetrahydrobiopterin dehydratase activities leads to the enhanced production of 7BH,.
Whether each or all of these metabolic aberrations are cause or conse- quence for the phenomenon vitiligo still remains to be identified.
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