E2 deficient mice accurately model classic MSUD
To demonstrate that the gene targeting event created a true E2 null allele, we determined BCKDH activity in liver homogenates of postnatal day 1 mouse pups derived from +/- by +/- mating pairs. As shown in Figure 2A, the BCKDH activity in +/+ mice was readily detectable. In marked contrast, BCKDH activity was completely absent in -/- mouse livers. As expected, homogenates from +/- mice had approximately half the activity of their +/+ littermates (Figure 2A). The results from -/- mice are similar to that observed in humans with classic MSUD [1].
Figure 2  Biochemical characterization of the classic MSUD murine model. A, BCKDH enzyme activity in liver of newborn wild type control (+/+), heterozygous (+/-), and homozygous (-/-) knockout mice. Enzyme activity was significantly reduced in +/- liver compared to +/+, and was below the level of detection in -/- liver. B, Total BCAA concentrations in blood of mice. Total BCAA represent the sum of leucine, isoleucine, and valine. Total BCAA concentrations in blood from -/- mice were significantly elevated compared to +/+ and +/-. C, Ratio of total BCAA to alanine in blood of mice. This ratio was significantly elevated in -/- mice compared to +/+ and +/- mice. The numbers on the bars indicates the number of mice analyzed. *, Significantly different from +/+ (P < 0.001); **, significantly different from +/+ and +/- (P < 0.001). Immunohistochemistry with an E2 specific antibody was used to examine E2 protein in the mice. As shown in Figure 1C and 1D, immunoreactive E2 protein was abundant in liver and embryonic fibroblasts of+/+ mice. In marked contrast, immunoreactive E2 protein was absent in these same tissues of -/- mice.
Homozygous E2 knockout mice had a nearly 3-fold increase in blood (Figure 2B) and urine (data not shown) levels of BCAA (sum of leucine, isoleucine, and valine) as compared to their +/+ littermates. Because amino acids were analyzed by tandem mass spectrometry, the sum of BCAA shown in Figure 2B also may include alloisoleucine that may have been produced in -/- MSUD mice.
The metabolism of BCAA is linked with the synthesis of alanine, glutamate, and glutamine [33]. Because of impaired metabolism of BCAA in MSUD mice, and to further characterize the abnormal biochemistry in this model, we analyzed the blood levels of the alanine, glutamate, and glutamine. As shown in Table 1, the levels of all three amino acids in homozygous mice were markedly lower than the levels in +/+ or +/- mice. The levels of these amino acids in the +/- mice were comparable to those in +/+ mice (Table 1). Because of the abnormal decrease in the blood alanine level and marked rise in blood BCAA levels that are characteristic of MSUD, a recent report on MSUD patients has suggested that the ratio of BCAA/alanine provides a more sensitive measure of the abnormal biochemistry of MSUD than BCAA level alone [8]. Therefore, we also expressed the amino acid results as BCAA/alanine ratio. As shown in Figure 2C, this ratio in -/- pups was more than 6-fold higher than +/+ or +/- littermates. These blood amino acid results are consistent with the concentrations seen in patients with MSUD [1,8,15].
Table 1  Summary of additional blood amino acid levels in the classic MSUD model and control littermates as determined by tandem mass spectrometry. All samples were collected on the day of birth. All values are mean +/- SEM.
MSUD Genotype  Alanine  Glutamate  Glutamine
(μmole/L)
+/+  196.1 ± 23.1 (6)  229.0 ± 12.0 (6)  76.8 ± 3.0 (6)
+/-  167.6 ± 9.9 (13)  251.0 ± 12.8 (13)  72.3 ± 1.6 (13)
-/-  94.6 ± 11.6* (5)  107.8 ± 4.8* (5)  50.4 ± 2.9* (5)
*: Significantly different from +/+ and +/- (P < 0.001). In summary, E2 knockout mice lack BCKDH enzymatic activity, E2 immunoreactivity, and have markedly elevated levels of BCAA in the blood and urine. These metabolic derangements ultimately result in neonatal lethality. These phenotypes are remarkably similar to that observed in humans with the classic form of MSUD. Thus, E2 knockout mice closely model classic MSUD.