Stabilization of uric acid mixed crystals by melamine

Abstract

Melamine stabilizes heterogeneous nucleation of calcium crystals by increasing the retention time and decreasing the rate of dissolution. Stabilization of such mixed crystals limit the efficacy of non-invasive treatment options for kidney stones. Crystalline forms of uric acid (UA) are also involved in urolithiasis or UA kidney stones; however, its interactions with contaminating melamine and the resulting effects on the retention of kidney stones remain unknown. Since melamine augments calcium crystal formation, it provides an avenue for us to understand the stability of UA-calcium phosphate (CaP) crystals. We show here that melamine facilitates UA+CaP crystal formation, resulting in greater aggregates. Moreover, melamine induced mixed crystal retention through a time-dependent manner in presence and/or absence of hydroxycitrate (crystal inhibitor), indicating its abridged effectiveness as conventional remedy. CaP was also shown to modify optical properties of UA+CaP mixed crystals. Differential staining of individual crystals revealed enhanced co-aggregation of UA and CaP. The dissolution rate of UA in presence of melamine was faster than its heterogeneous crystallization form with CaP, although the size was comparatively much smaller, suggesting disparity in regulation between UA and CaP crystallization. While melamine stabilized UA, CaP and mixed crystals in relatively physiological conditions (artificial urine), the retentions of those crystals were further augmented by melamine, even in presence of hydroxycitrate, thus reducing treatment efficacy.

Keywords: A1. Biocrystallization; A2. Heterogeneous Nucleation; B1. Solubility; B2. Kidney stones dissolution; B3. Calcium compounds.

Figure 1.

Uric acid and calcium phosphate (UA+CaP) crystals were formed with (A) 0 mM melamine (Mel), (B) 2.1 mM Mel, (C) 4.2 mM Mel, or (D) 6.25 mM Mel. Blue (UA), purple (CaP), and red (Mel) arrows are depicted in the images. (E) Relative crystal sizes were quantified using ImageJ and were represented as a bar diagram for mean+SEM. *, P<0.05. Scale bar (white) = 50 μm.

Figure 2.

(A) Time-dependent dissolutions of calcium phosphate (CaP), uric acid (UA), monosodium urate (MSU), UA+CaP, or MSU+CaP crystals in diH₂O at (B) 1 min, (C) 3 min, and (D) 10 min timepoints. (E) Time-dependent dissolutions of MSU+CaP and melamine (MSU+CaP+Mel) or UA+CaP+Mel crystals in diH₂O at (F) 1 min and (G) 3 min timepoints. (H) Time-dependent dissolutions of UA+CaP, MSU+CaP, UA+CaP+Mel, or MSU+CaP+Mel crystals with hydroxycitrate (HCit) in diH₂O at (I) 1 min and (J) 3 min timepoints. Bar diagrams depicting relative crystal sizes are represented in mean+SEM. *, P<0.05; **, P<0.01. Relative crystal sizes were measured using ImageJ software.

Figure 3.

(A) Calcium phosphate (CaP) and uric acid (UA) crystal formations were imaged with brightfield and polarized light microscopy after implementing UA (0, 2.1, 4.2, or 6.25 mM). (B) ImageJ measurements of relative crystal sizes are represented as a bar diagram in mean+SEM. *, P<0.01. Scale bar = 10 μm.

Figure 4.

(A) Time-dependent dissolutions of uric acid (UA) and/or calcium phosphate (CaP) crystals in with melamine (Mel) and/or hydroxycitrate (HCit) at (B) 1 min, (C) 3 min, and (D) 10 min timepoints in artificial urine. Bar diagrams depicting relative crystal index are represented in mean+SEM. **, P<0.01. Relative crystal sizes were measured using ImageJ software.