STUDY ON A NOVEL STRATEGY TO TREATMENT OF PHENYLKETONURIA

Figures & data

Figure 1. Novel strategy to treatment of PKU.

Figure 2. Construction of pMG36e PAL.

Table 1. The Blood Phe Concentration of All Rats on Different Days During Treatment

	1st Day		4th Day		9th Day		13th Day
	Control	Therapy	Control	Therapy	Control	Therapy	Control	Therapy
1	2.427	1.400	8.846	8.927	3.454	1.610	3.317	1.432
2	2.597	1.579	15.577	/	/	/	/	/
3	0.792	2.641	14.288	13.456	5.750	3.732	3.556	/
4	2.391	2.013	17.228	14.227	11.174	/	3.722	/
5	2.156	2.462	21.986	15.920	6.627	6.553	4.031	1.872
6	1.791	1.813	11.339	17.780	6.541	4.304	4.267	1.796
7	1.603	1.776	14.757	11.823	10.155	4.130	3.191	1.453
8	2.761	2.158	10.396	11.120	7.373	6.335	3.613	2.150
9	2.180	2.452	11.733	9.080	3.876	6.001	3.749	1.656
10	2.181	2.451	/	11.000	/	2.274	/	1.663
11	2.149	2.719	/	12.051	/	2.492	/	1.926
12	2.249	1.807	13.223	12.322	5.156	3.902	3.161	2.365
13	2.700	2.599	15.373	22.307	6.250	4.921	4.053	1.876
14	2.745	2.872	27.230	/	7.108	/	3.778	/
15	/	2.924	15.087	11.901	/	4.814	/	1.179
Average	2.141	2.310	15.168	13.225	6.679	4.256	3.676	1.750
value±SD	±0.548	±0.442	±4.915	±3.680	±2.337	±1.591	±0.358	±0.333
P value	P=0.393		0.266		0.008		0.000
	no significant difference		no significant difference		significant difference		significant difference

Unit::mg/100 ml.

Note: Wistar rats, divided into 2 groups (15 rats in each group), were used.(1) PKU therapy group: On 1–3 days P-CP/Phe solution was injected once a day and engineering L.L. containing pNZ(8048-PAL)1(GM17+PBS) was fed (2 ml/each) once a day; On 4–12 days stop injecting P-CP/Phe and fed the same engineering L.L (containing 25 mg/ml Phe)(2 ml/each).(2) PKU control group: The same scheme with PKU therapy group. Only one difference, that is, engineering L.L containing pNZ(8048-PAL)1 was replaced by engineering L.L. containing pNZ8048.

On the 1st, 4th, 9th, 13th day blood was drawed. The blood Phe concentration was measured by using HPLC.

Figure 3. PCR products from 8 clones of pMG36eaPAL/L.L. M: λDNA/HidIII digest.

Figure 4.

Figure 5. Analysis of PAL specificity in pET23bPAL/JM109DE3 by using HPLC. B1, B2 and B3 were from same clone pET23bPAL/JM109DE3. The only different between them was in culture medium: B1: NZCYM+0.1 mM Phe; B2: NZCYM+0.1 mM tyrosine; B3 NZCYM+0.1 mM Phe+0.1 mM tyrosine. Coumaric acid, the TAL deamination product from tyrosine, was not found in the three culture media above. Only Cinammic acid, the PAL product from Phe, was detected. The result shows: there is no TAL activity in the PAL product of pET23bPAL/JM109DE3, i.e. the specificity of the PAL is ideal.

Figure 6. Phe concentration in blood of the hyperphenylalanemia rats treated with two different preparations. *There is a significant difference between treatment group and control group (P<0.05).

Figure 7. Coomassie blue-stained gels after SDS-PAGE of extracts L.L. containing pNZ(8048-PAL)1 or pNZ(8048-PAL)2 producing PAL protein. Lane 1, molecular weight marker (in kilodaltons); Lane 2, uninduced pNZ(8048-PAL)1/L.L. cells; Lane 3, induced pNZ(8048-PAL)1 cells; Lane 4, uninduced pNZ(8048-PAL)2/L.L. cells; Lane 5, induced pNZ(8048-PAL)2/L.L. cells; Lane 6, uninduced pNZ(8048-PAL)2/L.L. cells; Lane 7, induced pNZ(8048-PAL)2/L.L. cells.

Figure 8. The blood Phe Concentration of each group on different days during treatment. Notes: (1) On the 1st day, blood Phe concentration was similar between two groups. (2) On the 4th day the Phe concentration in the therapy group was slightly lower than control group, but there was no significant difference between two groups. (3) On the 9th day the Phe concentration in the therapy group was distinctly lower than control group. There is significant difference between two groups (P=0.008<0.05). (4) On the 13th day the Phe concentration in the therapy group was 2 times lower than control group. There was extremely significant difference between two groups (P=0.000<0.01). The result shows that feeding engineering L.L. containing PAL gene can reduce the blood Phe concentration of PKU model rats.