Enhanced Activities of Blood Thiamine Diphosphatase and Monophosphatase in Alzheimer's Disease

Background Thiamine metabolites and activities of thiamine-dependent enzymes are impaired in Alzheimer’s disease (AD). Objective To clarify the mechanism for the reduction of thiamine diphosphate (TDP), an active form of thiamine and critical coenzyme of glucose metabolism, in AD. Methods Forty-five AD patients clinically diagnosed and 38 age- and gender-matched control subjects without dementia were voluntarily recruited. The contents of blood TDP, thiamine monophosphate (TMP), and thiamine, as well as the activities of thiamine diphosphatase (TDPase), thiamine monophosphatase (TMPase), and thiamine pyrophosphokinase (TPK), were assayed by high performance liquid chromatography. Results Blood TDP contents of AD patients were significantly lower than those in control subjects (79.03 ± 23.24 vs. 127.60 ± 22.65 nmol/L, P<0.0001). Activities of TDPase and TMPase were significantly enhanced in AD patients than those in control subjects (TDPase: 1.24 ± 0.08 vs. 1.00 ± 0.04, P < 0.05; TMPase: 1.22 ± 0.04 vs. 1.00 ± 0.06, P < 0.01). TPK activity remained unchanged in AD as compared with that in control (0.93 ± 0.04 vs. 1.00 ± 0.04, P > 0.05). Blood TDP levels correlated negatively with TDPase activities (r = -0.2576, P = 0.0187) and positively with TPK activities (r = 0.2426, P = 0.0271) in all participants. Conclusion Enhanced TDPase and TMPase activities may contribute to the reduction of TDP level in AD patients. The results imply that an imbalance of phosphorylation-dephosphorylation related to thiamine and glucose metabolism may be a potential target for AD prevention and therapy.


Introduction
3) Those with chronic alcohol abuse; 4) Those with decreased folate and vitamin B12 levels, and thyroid function. The demographic information was showed in Table 1.

Clinical assessments
All subjects were assessed general cognitive function with the Mini-Mental State Examination (MMSE, Chinese version). Control subjects without dementia were identified according to MMSE scores adjusted with years of education [21]: illiterate individuals with MMSE scores >17, subjects with a primary school diploma and scores >21, and subjects with >9 years of education and scores >24. All subjects were tested for levels of blood hemoglobin, liver and kidney functions. AD patients were further received neurological examination and neuropsychological evaluation including Activity of Daily Living (ADL) and Clinical Dementia Rating (CDR) scales by enquiring patients and his/her healthcare givers. AD patients were examined by cranial MRI and/or CT scanning and assessed levels of blood folate, vitamin B12, and thyroid hormones.
Determination of blood thiamine, TMP, and TDP The procedure was described in details previously [17]. Briefly, 150 μl of blood samples anticoagulated with heparin were collected and immediately deproteinized with 150 μl of 7.4% perchloric acid. The 300 μl of mixture was centrifugated at 10000 rpm for 6 min at 4˚C, and then the supernatant was collected and stored at −20˚C until use. Thiamine and its phosphate esters were derived into thiochromes using potassium ferricyanide and separated by gradient elusion with a C18 reversed-phase analytical column (250×4.6 mm). The derivatives were measured by HPLC fluoroscopy (Agilent 1100, Santa Clara, CA) with an excitation wavelength of 367 nm and an emission wavelength of 435 nm. Blood TDP, TMP and thiamine levels were quantified using standard samples of TDP, TMP, and thiamine (Sigma-Aldrich, St. Louis, MO).

Assay of blood TDPase, TMPase, and TPK activities
The activities of blood TDPase and TPK were determined as previously described with slight modification [11]. Briefly, 200 μl of EDTA-anticoagulated blood samples were homogenized with Tris-HCl buffer (20 mM, pH 7.4 containing 2 mM β-mercaptoethanol and 1 mM EDTA) in a Teflon-glass homogenizer. After the homogenate was centrifugated at 15,000 g for 40 min at 4˚C, the supernatant was transferred for TPK activity assay, whereas the pellet was suspended in 225 μl of Tris buffer (50 mM, pH 7.4 containing 1% Triton X-100) for TDPase activity assay. To assay TDPase activity, 360 μl of Tris buffer (50 mM, pH 7.4containing 5.5 mM MgCl 2 ) was added to 100 μl of the pellet fraction. The reaction was initiated by adding 40 μl of TDP (2.5 μM, pH 7.4) at 37˚C and terminated after 30 min incubation by adding 500 μl of perchloric acid (5.4%). The levels of TDP, TMP, and thiamine were then determined by HPLC. TDPase activity was calculated according to the generation of TMP content (nmol) per mg protein per minute.
To assay TPK activity, 280 μl of Tris-HC1 (180 mM, pH 7.5 containing 64 mM MgSO4) and 60 μl of ATP (500 mM, neutralized to pH 7.5) were added to 100 μl of the supernatant. The reaction was initiated by adding 100 μl of thiamine (1.0 μM), and terminated after 1 h incubation at 37˚C by adding 540 μl of perchloric acid (5.4%). After the proteins were sedimented (10,000 g, 15 min), the levels of TDP, TMP, and thiamine in the supernatant were then measured by HPLC. TPK activity was represented as the generation of TDP content (nmol) per mg protein per minute.
The assay of TMPase activity was conducted according to Rao's protocol with slight modification [22]. Briefly, 200 μl of EDTA-anticoagulated blood samples were homogenized with 1 ml of 0.32 M sucrose containing 1% Triton X-100. Then the homogenates were centrifuged at 1,000 g for 10 min at 4˚C, and the supernatant was collected for TMPase activity assay.
To assay TMPase activity, 1 ml of Tris-maleate buffer (150 mM, pH 5.5, containing 4 mM MgCl 2 ) was mixed with 100 μl of the supernatant and pre-incubated for 5 min at 37˚C. The reaction was initiated by adding 100 μl of TMP (4.0 μM), and terminated after 1 h incubation by adding equivoluminal perchloric acid (5.0%). After the proteins were sedimented (10,000 g, 15 min), the levels of TDP, TMP, and thiamine in the supernatant were then measured by HPLC. TMPase activity was calculated according to the generation of thiamine content (nmol) per mg protein per minute.
Protein concentrations were determined with the Pierce™ BCA protein assay kit according to the manufacturer's instructions (thermo scientific, Rockford, IL). All the enzyme activities were assayed concurrently for parallel groups of AD and control subjects, and the activity values for individual AD patients (%) were normalized to the average value of control group (100%) during the same assay. Enzyme activities were repeated twice and an average value was obtained.
Apolipoprotein E (APOE) genotypes analysis APOE allele analysis was conducted using an ABI real-time Taqman SNP genotyping assay (ABI, Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Genomic DNA was purified from blood by using a genome extraction kit (TIANGEN, Beijing, China). Genomic DNA was used for allelic determination of SNP 112 (rs429358: GCCCCGGCCTG GTACAC) and SNP 158 (rs7412: GGCACGGCTGTCCAAGGA) of APOE gene. 1 μl of DNA is combined with 10 μl of 1 X final concentration of universal Taq Man PCR Master mix, 8.75 μl of sterilized water and 0.25 μl of primers for SNP 112 (rs429358) or SNP158 (rs7412). Both SNP assays were done separately on 2 plates for the same samples, the results were recorded and combined for genotypes determination.

Statistical Analysis
SPSS software (version 18.0; SPSS Inc) was used for statistical analyses. Mann Whitney U test or Student t-test (two-tailed) and Chi-square test were used to compare demographic data among control subjects and AD patients. Kruskal-Wallis test and Mann Whitney U test were used to compare data among subgroups of control subjects and AD patients. All data are shown as the mean± SEM except for noted.

Characterization of study participants
The demographic data are shown in Table 1 (mean ± SD). There is no significant difference in age, gender, or years of education between AD patients and control subjects (P = 0.514 for gender, P = 0.736 for age, and P = 0.462 for years of education). AD patients had significantly lower MMSE scores than control subjects (5.62 ± 6.28 vs. 27.08 ± 2.24, P < 0.0001). Blood TDP levels were significantly decreased in AD patients as compared with that in control subjects (79.03 ± 23.24 vs. 127.60 ± 22.65nmol/L, P < 0.0001) whereas TMP levels showed a trend to increase in AD patients as compared with control subjects, but the change did not reach statistical significance (5.33 ± 4.44 vs .3.81 ± 2.91nmol/L, P = 0.077). Neither was there significant difference in thiamine levels between the two groups (3.62 ± 3.87 vs. 3.95 ± 3.59 nmol/L, P > 0.05). Twenty-four of AD patients (24/45, 53.33%)were carriers of APOE ε4 allele (21 patients with 3/ 4 genotype, 3 with 4/4 genotype). The proportion of APOE ε4 allele carriers in AD patients was significantly higher than that in control subjects (3/38, 7.89%, 2 subjects with 2/4 genotype and 1 with 3/4 genotype; P < 0.0001). The levels of fasting plasma glucose in AD patients were significantly lower than that in control subjects (5.43 ± 1.52 vs. 6.13 ± 1.51mmol/L, P < 0.05).
Further analysis was performed on the correlation between TDP and TMP contents. No significant correlation was observed in all participants or in AD patients. However, a positive correlation was observed in control subjects (S2 Fig). Effects of fasting glucose levels on blood TDPase, TMPase, TPK activities  Effects of the disease severity, age, and APOE ε4 allele on blood TDPase, TMPase, and TPK activities To clarify the effect of disease severity on blood TDPase, TMPase, and TPK activities, AD patients were divided into different subgroups according to their MMSE (severe subgroup: MMSE<10, mild to moderate subgroup: MMSE ! 10) or ADL scores (severe subgroup: ADL<23, mild to moderate subgroup: ADL ! 23). We divided AD patients into three subgroups according to their CDR scores (severe subgroup: CDR = 3, moderate subgroup CDR = 2, mild subgroup CDR 1). No significant differences were found in TDPase activities in all subgroups based on MMSE (severe subgroup: 1.24±0.11, mild to moderate subgroup: 1.

Discussion
Previous studies demonstrated that thiamine deficiency can drive AD-like pathophysiological alterations. Decreased TDP levels and thiamine-dependent enzymes activities were found in AD patients but not in patients with vascular dementia, frontotemporal dementia and Parkinson's disease [9,16]. Our previous study demonstrated that thiamine metabolism can serve as a promising biomarker for AD diagnosis with high sensitivity and specificity over 80% [8]. However, the mechanism for impaired thiamine metabolism in AD need to be clarified.
In this study, we demonstrated for the first time that blood TDPase and TMPase activities in AD patients were significantly enhanced as compared with those age-and gender-matched control subjects whereas TPK activity was not significantly changed (Fig 1). Although only TDPase and TPK, but not TMPase, activities significantly correlated with TDP levels in all subjects (Fig 2), the reduction of blood TDP levels in AD patients (Table 1) should still be attributed to the enhanced dephosphorylation due to elevated activities of TDPase and TMPase. Further, we analyzed the correlation between TDP and TMP levels respectively in AD patients and control subjects. We found that a significantly positive correlation in control subjects but not in AD patients (S2 Fig), which suggested that the balance between TDP and TMP contents was disrupted due to the enhanced TDPase and TMPase activities in AD patients. Since the three TDP-dependent enzymes, PDH, KGDH and TK, are critical for glucose metabolism, our current results strongly suggest that an impairment of phosphorylation and dephosphorylation in thiamine metabolism contributes to TDP reduction and, thus, brain glucose hypometabolism in AD.
We also investigated the association between TDPase, TMPase, and TPK activities and the disease severity evaluated by MMSE, CDR, and ADL scores in AD. Our analysis showed no significant correlations between disease severity or age and TDPase, TMPase, and TPK activities ( S3 Fig and S4 Fig). Aging is a major risk factor for AD. Besides, the elderly tend to have thiamine deficiency [23,24]. However, thiamine supplementary or high thiamine dietary had a weak effect on prevention of cognitive decline with aging [25]. The finding that activities of two thiamine-metabolizing phosphatases are enhanced independent of the disease severity and age implies that the imbalance between phosphorylation and dephosphorylation of thiamine metabolism in AD is involved in pathophysiology of the disease itself rather than other illness-related changes, such as the life style and disease duration. In addition, we also excluded the possible effects of fasting plasma glucose level and APOE ε4 allele on the enzymatic activities of TDPase, TMPase, and TPK. The results showed that there were no differences in TDPase, TMPase, and TPK activities between APOE ε4-carriers and non-ε4-carriers in both control subjects and AD patients. Also, fasting plasma glucose level did not significantly correlate with blood TDPase, TMPase, and TPK activities (S4 Fig & Fig 3). Our data showed that blood TDPase activity increased by 24%, TMPase activity increased by 22% whereas TPK activity remained stable in AD patients as compared with those in control. The results differ from two previous studies [10], which showed either significantly decreased or unchanged activities of TDPase and TMPase in AD. The differences might be due to the different tissue types and the sample sizes used by the studies. The previous studies measured the enzymatic activities of TDPase and TMPase using autopsied brain tissues. The large variation in the time span from death to brain tissue collection could impact the sample quality for the measurement of enzymatic activities. Previous studies suggested that thiamine metabolizing enzymes and transporters showed different expression patterns of different organs [26,27]. Our unpublished data also showed different TPK and TDPase activities in brain, kidney, liver, blood cells and plasma of mice. Hence, sample types may be a crucial factor for enzymatic activities assay. In addition, the small sample sizes used in the previous studies could also affect the accuracy of the results.
AD pathogenesis is still unclear. Perturbed cerebral glucose metabolism is an important pathophysiological feature and precedes the onset of symptoms in AD [2,28,29,30]. Since TDP is a critical coenzyme for glucose metabolism, our study suggests that elevated TDPase and TMPase activities that lead to the reduction of TDP may contribute to brain glucose hypometabolismin AD. In addition, a previous study has demonstrated that TMPase is present only in glial enriched fractions, whereas TDPase is 20.8-fold higher in neuronal than in glial fractions [31]. It can be reasonably presumed that elevated activities of the two phosphatases originated mainly from different cell types are anon-specific consequence secondary to other AD pathophysiological alteration(s). Thus, it is possible that activities of other phosphatases are also increased in AD. Indeed, increased activities of serum ACP and ALP were observed by Vardy E and Kellete K's studies [32]. The enhancement of non-specific phosphatase activities may be the main reason for the imbalance between phosphorylation and dephosphorylation in AD [33]. For example, increased activities of phosphatases induced dephosphorylation of glucogen synthase kinase-3 and, consequently, lead to the augmentation of its activity and tau hyperphosphorylation. Inhibiting phosphatases may be a potential target for AD prevention and treatment.

S4 Fig. Correlations between blood TDPase, TMPase, TPK activities and ages in AD patients, control subjects or all subjects. (A).
There was no significant correlation between age and TDPase activities in AD patients (n = 45), control subjects (n = 38) or all participants (n = 83). (B). No significant correlation was observed between age and TMPase activities in AD patients, control subjects or all participants. (C). There was no significant correlation between age and TPK activities in AD patients, control subjects or all participants. (TIF) S5 Fig. Effects of APOE ε4 allele on blood TDPase, TMPase and TPK activities. (A). There was no significant difference in TDPase activities between APOE ε4 allele carriers (n = 24) and non-carriers in AD patients. (B). No significant difference was observed in TMPase activities between APOE ε4 allele carriers and non-carriers in AD patients. (C). There was no significant difference in TPK activities between APOE ε4 allele carriers and non-carriers in AD patients. (D). There was no significant difference in TDPase activities between APOE ε4 allele carriers (n = 3) and non-carriers in control subjects (n = 35). (E). No significant difference was observed in TMPase activities between APOE ε4 allele carriers and non-carriers in control subjects. (F). There was no significant difference in TPK activities between APOE ε4 allele carriers and non-carriers in control subjects. (TIF)