Effects of Alzheimer-Like Pathology on Homocysteine and Homocysteic Acid Levels-An Exploratory In Vivo Kinetic Study

Abstract

Hyperhomocysteinemia has been suggested potentially to contribute to a variety of pathologies, such as Alzheimer's disease (AD). While the impact of hyperhomocysteinemia on AD has been investigated extensively, there are scarce data on the effect of AD on hyperhomocysteinemia. The aim of this in vivo study was to investigate the kinetics of homocysteine (HCys) and homocysteic acid (HCA) and effects of AD-like pathology on the endogenous levels. The mice received a B-vitamin deficient diet for eight weeks, followed by the return to a balanced control diet for another eight weeks. Serum, urine, and brain tissues of App^NL-G-F knock-in and C57BL/6J wild type mice were analyzed for HCys and HCA using LC-MS/MS methods. Hyperhomocysteinemic levels were found in wild type and knock-in mice due to the consumption of the deficient diet for eight weeks, followed by a rapid normalization of the levels after the return to control chow. Hyperhomocysteinemic App^NL-G-F mice had significantly higher HCys in all matrices, but not HCA, compared to wild type control. Higher serum concentrations were associated with elevated levels in both the brain and in urine. Our findings confirm a significant impact of AD-like pathology on hyperhomocysteinemia in the App^NL-G-F mouse model. The immediate normalization of HCys and HCA after the supply of B-vitamins strengthens the idea of a B-vitamin intervention as a potentially preventive treatment option for HCys-related disorders such as AD.

Keywords: alzheimer disease; animal; disease models; hyperhomocysteinemia; vitamin B deficiency.

Figure 1

Serum homocysteine levels in C57BL/6J wild type and App^NL-G-F knock-in mice, data are depicted for males and females separately for experimental week 1-17: baseline measurement (w. 1), diet deficient in vitamin B6, B12 and folate (for 8 w.), balanced normal chow (for 8 w.); all serum samples were analyzed using a combination of liquid chromatography with tandem mass spectrometry and statistically tested non-parametrically using the Kruskal-Wallis test; data are presented as median ± interquartile range (IQR); extreme outliers beyond 3xIQR have been excluded.

Figure 2

Serum homocysteic acid levels in C57BL/6J wild type and App^NL-G-F knock-in mice, data are depicted for males and females separately for experimental week 1–17: baseline measurement (w. 1), diet deficient in vitamin B6, B12 and folate (for 8 w.), balanced normal chow (for 8 w.); all serum samples were analyzed using a combination of liquid chromatography with tandem mass spectrometry and statistically tested non-parametrically using the Kruskal-Wallis test; data are presented as median ± interquartile range (IQR); extreme outliers beyond 3xIQR have been excluded.

Figure 3

Urinary homocysteine levels in C57BL/6J wild type and App^NL-G-F knock-in mice, data are depicted for males and females separately for experimental week 1-17: baseline measurement (w. 1), diet deficient in vitamin B6, B12 and folate (for 8 w.), balanced normal chow (for 8 w.); all urine samples were analyzed using a combination of liquid chromatography with tandem mass spectrometry and statistically tested non-parametrically using the Kruskal-Wallis test; data are presented as median ± interquartile range (IQR); extreme outliers beyond 3xIQR have been excluded.

Figure 4

Urinary homocysteic acid levels in C57BL/6J wild type and App^NL-G-F knock-in mice, data are depicted for males and females separately for experimental week 1–17: baseline measurement (w. 1), diet deficient in vitamin B6, B12 and folate (for 8 w.), balanced normal chow (for 8 w.); all urine samples were analyzed using a combination of liquid chromatography with tandem mass spectrometry and statistically tested non-parametrically using the Kruskal-Wallis test; data are presented as median ± interquartile range (IQR); extreme outliers beyond 3xIQR have been excluded.

Figure 5

Homocysteine levels in brain tissue in C57BL/6J wild type and App^NL-G-F knock-in mice, data are depicted for males and females separately for experimental week 1–17: baseline measurement (w. 1), diet deficient in vitamin B6, B12 and folate (for 8 w.), balanced normal chow (for 8 w.); all brain samples were analyzed using a combination of liquid chromatography with tandem mass spectrometry and statistically tested non-parametrically using the Kruskal-Wallis test; data are presented as median ± interquartile range (IQR); extreme outliers beyond 3xIQR have been excluded; ~indicates levels below the lower limit of quantification (LLOQ).

Figure 6

Homocysteic acid levels in brain tissue in C57BL/6J wild type and App^NL-G-F knock-in mice, data are depicted for males and females separately for experimental week 1–17: baseline measurement (w. 1), diet deficient in vitamin B6, B12 and folate (for 8 w.), balanced normal chow (for 8 w.); all brain samples were analyzed using a combination of liquid chromatography with tandem mass spectrometry and statistically tested non-parametrically using the Kruskal-Wallis test; data are presented as median ± interquartile range (IQR); extreme outliers beyond 3xIQR have been excluded.

Figure 7

Time line of the study course, diet regimen and sampling points: S = serum sampling, U = 24-h urine sampling in metabolic cages, B = brain sampling: one half of the animals was euthanized in the middle (experimental week 9) and the other half at the end of the study (experimental week 17); feeding a diet deficient in vitamin B6, folate (B9) and B12 for 8 weeks was followed by a normal diet period for another 8 weeks.