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Analysis of the enzyme activity expressed as rate of product
Analysis of the enzyme activity, expressed as rate of product formation per unit serum volume (20μl), between the diagnostic groups revealed that ApB activity was significantly decreased in the patient group both at recruitment and after 13months when the substrate concentration used was 1.5mM (p<0.05 in both cases). When the substrate concentration was 3.0mM, the Nimodipine in ApB activity became non-significant (p=0.2839). Activities of ApA and ApN were significantly reduced in the patient group, at the 13month time point only, when the substrate concentration was 1.5mM (p<0.05 in both cases, Fig. 2). There were no significant differences in IRAP activity between the groups.
Serum protein concentrations were determined for the control group and the second (13month) patient group only. Analysis of enzyme activity, determined as product formation (mMol) per minute per mg serum protein demonstrated that, at a substrate concentration of 1.5mM, only ApB had significantly decreased enzyme activity in the Alzheimer's disease group (p<0.05). At a substrate concentration of 3mM, only ApA showed significantly reduced activity in the patient group (p<0.05, Fig. 3).
A more elegant analysis of the enzyme activity is achieved by fitting same results for the rate of product formation at different substrate concentrations to the Michaelis-Menten equation, see Fig. 4 for an illustrative example based on the results for ApB displayed in Fig. 2. Such analysis allows estimation of values for maximal rate of product formation (Vmax) and the substrate concentration at 50% Vmax (Michaelis-Menten constant, KM.
Table 2 gives the estimated enzyme kinetic parameters for the ApB, ApN and IRAP; changes in activity of ApA with substrate concentration were such that it was not possible to estimate values for Vmax or KM with any accuracy. One-way ANOVA was unable to detect any significant differences in the parameters for ApB, ApN or IRAP between the diagnostic groups.
Despite there being some evidence of significantly decreased aminopeptidase activity in patients with AD, with some changes over time/severity of the disease, there were no significant correlations between activity of any of the enzymes studied (at substrate concentrations of 3mM) and neuropsychological test scores (Pearson product moment correlation).
Discussion
Puertas et al. (2013), and to some extent Kuda et al. (1997) showed that plasma aminopeptidase activity was decreased in AD; this study sought to replicate and explore potential mechanisms underlying the observed changes. The current study identified reduced activities of ApA, ApB and ApN within the AD population, but unlike the work of Puertas and colleagues, no difference in IRAP activity. The decreased activities identified were dependent on substrate concentration and to some extent disease severity.
One of the possible confounding factors between studies was the ages of the patient and control groups. In the study of Puertas et al. (2013) the two groups were very closely matched, whilst in the current study the controls were approximately 6years younger than the AD patients; Kuda et al. state that the groups were age matched without giving any ages. Such an age difference in the current study may have obscured any differences in enzyme activity between the groups as there is evidence of increasing aminopeptidase activity with age (e.g. Ijima et al., 2002); any such age-related increase in enzyme activity might explain the failure to identify reduced aminopeptidase activity the older AD patients. Although the patients of the current study were markedly older than those studied by Puertas et al. (2013), by approximately 8years, the MMSE scores (derived from ACE-R), however, were very similar, suggesting a comparable disease severity. Unlike the study of Puertas, the current study did not detect any significant relationship between enzyme activity and MMSE score, nor scores on any of the other, more sensitive, psychometric tests. MMSE is recognised as a coarse measure of cognitive ability (Votruba et al., 2016) and thus lack of any robust association with enzyme activity is not surprising. The independence of minor variabilities in scores on the other psychomotor tests and enzyme activity underlines the fact that cognitive function and plasma aminopeptidase activity are probably not precisely nor causatively associated.