A novel method for quantifying peripheral tissue amyloid load by using the radiolabeled amyloidophilic peptide, p5

Abstract

Quantitation of peripheral amyloid deposits by non-invasive molecular imaging can be useful for diagnosis, prognostication and monitoring response to therapy. In order to obtain reliable quantitative data, it is necessary to show a linear positive correlation between the uptake of the molecular probe and the tissue amyloid load. The transgenic H-2/IL-6 mouse model of AA amyloidosis was used to generate animals with varied stages of visceral amyloid disease. The mice were injected with 125I-labeled peptide p5 and tissues analyzed 2 h post-injection using Congo red (CR) staining, radioisotope biodistribution and micro-autoradiography (ARG). Micro-ARG confirmed that 125I-p5 was deposited at all amyloid deposits and sites of Congophilia but not at amyloid-free sites within the tissues evaluated. Furthermore, biodistribution studies revealed that the amount of 125I deposited in liver and spleen correlated with the amount of CR birefringence (expressed as 0-4+ or as tissue area [µm2]) in these tissues with correlation coefficients of r > 0.7 (p < 10(-6)). Deposition of 125I-p5 is a quantitative measure of the amount of AA amyloid in liver and spleen in this mouse model. The p5 peptide has potential as a quantitative amyloid imaging agent in human disease.

Figure 1

Radiolabeled peptide p5 binds AA amyloid in vivo. The reactivity of the ¹²⁵I-p5 peptide in stomach liver and kidney was evidenced by the appearance of black deposits in micro-autoradiographs (ARG) that correlated with the distribution of Congo red (CR)-birefringent amyloid in consecutive tissue sections.

Figure 2

Quantification and ¹²⁵I-p5 labeling in splenic AA amyloid in mice. ¹²⁵I-p5 was seen in micro-autoradiographs (ARG) to bind weak (1+) and intense (4+) splenic AA amyloid . The ¹²⁵I-p5 distribution correlated with Congo red (CR)-birefringent amyloid that was then quantitatively measured by image analysis (CRq – area detected by algorithm is false colored red).

Figure 3

The binding and retention of ¹²⁵I-p5 peptide in the liver and spleen correlates with amyloid load. Linear regression analysis with 95% confidence lines (dotted) or ¹²⁵-p5 (% injected dose/gram tissue) and Congo red scoring (0 – 4+: CRsq) and Congo red area on the stained tissue (CRq).

Figure 4

Splenic AA amyloid load correlates positively with deposits in the liver in H-2/IL-6 transgenic mice. Amyloid burden was measured by ¹²⁵I-p5 peptide retention in vivo (%ID/g), Congo red scoring (0 – 4+: CRsq), and measuring Congo red-birefringent area (µm²: CRq).