On the basis of clinical data (Table ), five patients corresponded to moderate PKU, and one patient had a severe type. Mutations were mostly typical for European patients with PKU, e.g., R408W, R261Q, G272X. In P225R, first described in a Sweden, and in Y166X, which was not described earlier, the severity of the mutations are unknown. In patient no. 5, one mutation could not be determined, despite repeated DGGE analysis. Assuming a large deletion or mutation in one of the large introns, a null mutation (residual activity <1%) is most probable. Regarding Phe tolerance at five years (12
) and data from standardized protein loading tests (13
), clinical phenotype and genotype corresponded. Neurologic status was normal except for mild tremor, which was present in two patients. Long-term baseline Phe values during adult age were lower in patients on diet compared with patients off diet.
Plasma Phe was normal in all control subjects (mean 66 ± 9 μmol/l). In the six patients with PKU, preload plasma Phe values (Table ) ranged between 796 and 1,459 μmol/l and were thus in the typical long-term range of these patients. Mean preload plasma Phe concentrations before start of the Phe load and LNAA treatment did not differ between both series (t = 0.76, not significant [ns]). Mean pretreatment plasma concentrations of all other LNAAs (Phe+LNAA –10 and –2.5 h; Pheonly –0.5 h) were also comparable for the two series (Table and Figure ). Before the Phe load, mean Km-normalized Phe ratios of 5.7 and 5.6 resulted for the Pheonly and the Phe+LNAA series, respectively (Table ), in comparison with a mean Km -normalized Phe ratio of 0.33 ± 0.04 in the control subjects. Preload Km-normalized Tyr and Trp ratios for the PKU patients were 0.015 and 0.026, respectively, in both series (Table ), compared with 0.09 (Tyr ratio) and 0.19 (Trp ratio) in the control subjects.
Figure 3 Time course of plasma and brain Phe and examples for LNAAs. (a) Steep increase of plasma Phe levels after an oral dose of L-Phe (time 0 h) in the Pheonly (dotted line) and Phe+LNAA series (solid line). (b) Plasma concentrations of valine and ( (more ...)
Plasma and brain Phe concentrations, Km-normalized Phe/LNAA ratio, plasma/brain Phe ratio, and EEG spectral parameters in PKU patients as functions of time
In all 12 preload 1H-MR spectra of the patients, excess Phe peaks at 7.37 ppm were identified; these peaks are attributed to the phenyl protons of Phe. Figure contains the spectrum for one patient before the Phe load in the Pheonly series (plasma Phe of 1,193 μM; brain Phe 265 μmol/kg) and demonstrates the signal-to-noise ratio achieved for single data points. Absolute brain tissue concentrations of Phe are listed in Table . Mean statistical uncertainty of a single brain Phe measurement (mean SEM of four spectra for each session) was 17 and 19 μmol/kg for the Pheonly and Phe+LNAA series, respectively, i.e. ~7% of the measured values. Preload brain Phe concentrations were not significantly different (t = 1.4, ns) for the two series. However, the mean plasma/brain ratio was somewhat lower during the Pheonly series (t = 2.05, P < 0.10). Preload plasma and brain Phe concentrations correlated significantly during the Pheonly series (τb = 0.87, P < 0.05) but not during the Phe+LNAA series (τb = 0.33, ns).
Dynamic data during Phe and LNAA intake.
After the oral Phe load, plasma Phe values (Figure a) steeply increased to reach a maximum one hour postload during both series and slowly decreased thereafter. In the Pheonly series, plasma Phe increase in comparison with preload values was 82% (854 ± 101 μmol/l) six hours postload, 78% (802 ± 133 μmol/l) 12 h postload, and 63% (657 ± 123 μmol/l) 24 h postload. In the Phe+LNAA series, plasma Phe increase was 75% (797 ± 368 μmol/l) six hours postload, 55% (579 ± 238 μmol/l) 12 h postload, and 38% (405 ± 212 μmol/l) 24 h postload. Comparing plasma Phe concentrations in the two series, these were similar six hours postload (t = 0.02, ns). In the Phe+LNAA series, plasma Phe was 9% lower 12 h postload (t = 2.5, P < 0.10) and 12% lower 24 h postload (t = 3.6, P < 0.05) in comparison with the Pheonly series. Phe levels had decreased by 17% (Pheonly) and 20% (Phe+LNAA), respectively, at 24 h compared with maximum Phe at one hour postload. Ten days later, Phe levels had returned to preload values in both series and in all patients.
During the Pheonly series, plasma concentrations of some other LNAAs decreased slightly in the postload period (Table ). This was statistically significant for isoleucine (t = 3.8, P < 0.05) and Trp (t = 6.7, P < 0.01). During the Phe+LNAA series, LNAA supplementation started at minus two hours and led to a substantial increase in plasma LNAA concentrations in all seven AAs (Table ). The courses of valine (Figure b), methionine, isoleucine, and leucine plasma concentrations from preload to 12 h postload showed a marked jitter, attributed to both fast absorption from the gastrointestinal tract and fast elimination from blood, whereas the courses of Tyr, histidine, and Trp (Figure c) concentrations were found to be smoother. During LNAA treatment, lysine concentrations decreased by ~30%. As lysine levels also slightly decreased in the Pheonly series compared with pretreatment levels (Table ), there was no significant difference between the series (t =1.9, P = 0.11). However, an effect of LNAA treatment on plasma lysine levels has to be assumed. Also, other AAs showed a slight decrease of plasma concentrations postload. This effect was present in both series, e.g., ornithine Pheonly series: –14 μmol/l, approximately –18%; Phe+LNAA series: –10 μmol/l, approximately –12%, compared with pretreatment levels. Most LNAA concentrations and lysine were back in the pretreatment range 14 h after stopping LNAA supplementation, i.e., 24 h after the Phe load. However, mean concentrations for methionine (+196%), Tyr (+43%), and histidine (+20%) were still above pretreatment levels. Ten days after the Phe load, all LNAA levels were in the pretreatment range.
The Km-corrected Phe/LNAA ratio (Table and Figure d) more than doubled in the Pheonly series, whereas it decreased under LNAA treatment, i.e., up to 12 h. In the Pheonly series, Km-normalized Tyr and Trp ratios decreased. However, in the Phe+LNAA series, they increased despite elevation of plasma Phe.
In vivo 1H-MRS (Figures e and ) revealed totally different courses of cerebral Phe levels with and without LNAA supplementation. The data from the Pheonly series confirmed that an increase in plasma Phe leads to a significant, but delayed, increase of Phe concentrations in brain tissue (preload to six hours postload; t = 6.4, P < 0.01). On average, this increase still continued after 12 h, and brain Phe levels were higher at 24 h compared with 12 h postload (Table ). Total increase from preload to 24 h postload was 64%. During the Phe+LNAA series, the influx of Phe into brain tissue was totally blocked for 12 h (Figure e). From preload to 12 h postload, brain Phe even decreased by 5%, although this difference was not significant (t = –1.2, ns). After stopping LNAA supplementation, brain Phe also increased during the Phe+LNAA series. Total increase from preload to 24 h postload was 41% in the Phe+LNAA series (t = 3.8, P < 0.05). The effects of Phe loading during the two series are also viewed in Figure . The differing sizes of the Phe peaks from difference spectra (PKU minus control) reflect the differing cerebral Phe levels. Because of a delayed influx from plasma into brain tissue, plasma/brain ratios of Phe (Figure f) increased during the Pheonly series from preload (4.0) to six hours (5.3) and 12 h (4.7) postload. This ratio returned to preload values 24 h postload (4.2). As plasma Phe concentrations increased, and brain Phe remained stable, the increase of the plasma/brain ratio six hours and 12 h postload was much more pronounced during the Phe+LNAA series.
Figure 4 Averaged 1H-MRS difference spectra (patients minus averaged normal spectra), acquired in vivo before as well as 6, 12, and 24 h after the oral Phe load. The increase of the Phe peak at 7.37 ppm during the Pheonly series (dotted line) contrasts with the (more ...)
The 1H-MR spectra did not show contributions from any of the supplemented LNAAs. In particular, there was no indication for a doublet at 1 ppm that would be characteristic for valine, which reached almost 1 mM in plasma.
Monitoring brain activity by EEG spectral analysis.
The effect of Phe intake on EEG activity (Table and Figure ) was also different for the two series. Although the individual profiles of the power spectra were reproduced in all EEG derivations of both series, a shift of the dominant peak of background activity to the lower-frequency spectrum was observed for the Pheonly series from six hours postload onward. This dominant peak was located in the center of the α band in five patients. Figure contains the averaged EEG power spectra of all six patients from –2.5 h preload to 24 h postload. Calculation of the relative power in conventional frequency bands confirmed Phe-related changes in EEG spectra. A significant increase of θ activity from preload to 24 h postload (t = 3.6, P < 0.05) was accompanied by a simultaneous decrease of α2 activity (t = –3.5, P < 0.05). Consequently, the α/θ ratio decreased continuously from preload to 24 h postload in the Pheonly series. Similarly, PF (t = –6.0, P < 0.01) and MPF (t = –4.9, P < 0.01) decreased by 0.4 Hz (Table ). These changes were noted in all six patients; δ, α1, and β activity were unchanged.
Averaged EEG power spectra from patients with PKU. During the Pheonly series (dotted line), a shift of the dominant peak of EEG background activity to the lower-frequency spectrum is demonstrated, which was prevented by LNAA treatment (solid line).
During the Phe+LNAA series, comparable effects were not observed (Figure ). EEG power spectra from –2.5 h preload (before first LNAA intake) and –0.5 h preload (after first LNAA intake) were comparable. An increase in fast α activity, as well as PF and MPF, was observed in EEGs 12 h postload, i.e., around midnight. The following morning, which was 24 h after Phe intake, EEG activity was not significantly different compared with preload measurements. Comparing Pheonly and Phe+LNAA series, differences were statistically significant for θ activity 24 h postload, α2 activity 12 and 24 h postload, as well as the α/θ ratio 12 and 24 h postload (Table ).
Visual evaluations of all EEG recordings from both series revealed no abnormalities. In particular, abnormal EEG patterns, like epileptiform activity, were not observed. During both Pheonly and Phe+LNAA series, no side effects were registered, except for feelings of satiation after LNAA intake. Blood glucose levels were above 4.4 mmol/l at regular checks. Standard laboratory evaluations (red and white blood cell counts, electrolytes, transaminases) remained in the normal range.