A new concept for Urate in human serum: Evidences that support the existence of Urate as loosely associated and protein-bound Urate in human serum

According to the fact that uric acid has its maximum absorbance in the U.V. light between the W.Ls of 290 nm and 295 nm under optimized conditions and making use of the specific destructive effect of uricase on uric acid, the absorption spectrum of a serum sample was carefully studied in the region between 270–300 nm before and after incubation with uricase.

The results obtained are illustrated in tables 1,2 and figures 1,2,3,4 &5.

Table 1 (a,b) & figure 1-curve a; denote a maximum decrease in absorbance at 283–290 nm after the action of uricase on the buffered diluted serum. On the other hand, the strongest band recorded for the pure uric acid solution was at 293 nm, table 1 { U₀-U₂) } & figure 1 curve-b; the same wave length at which urate in the buffered protein-free clear supernatant also gives its maximum peak., Table 2 (A₀ - A₂.), figure 2b.

In addition, table 2, B₀ - B_24, & figure 2a; show a maximum decrease in absorbance at 283-285 nm when the buffered protein solution was left under the action of uricase for 24 hours.

After longer incubation periods, 48 hours, table 1, C & table 2, B₇₂, the recorded absorbance was higher than the original values obtained before the action of uricase, table 1, A & table, B₀.

The absorption spectra of the clear protein-free supernatant after subjecting the serum for the action of sodium hydroxide solution 0.4 mol/L at 60°C for 5, 10 and 15 minutes, before and after destruction with uricase, as presented in figures 3,4 and 5 showed maximum decrease in absorbace at 295 nm – the same W.L at which the free uric acid gives its maximum absorption peak –, along the experiment periods of time (5,10 & 15 minutes),

Moreover, after 15 minutes, more urate were liberated and absorb at 290 nm – figure 5.

The results showed a remarkable difference in the wave lengths (W.Ls) at which urate in serum gives its maximum absorbance, 283–290 nm, compared to 293 nm, the W.L. at which the pure uric acid solution gives its maximum band.

This, could be attributed to the presence of a sort of co-ordination between serum urates and proteins under physiologic conditions. As soon as the proteins were precipitated, part of the serum urates were identified in the protein-free supernatant at the same wavelength at which the free uric acid solution has its maximum peak. This part of serum urate is easily attacked by uricase.

The other part of serum urate appeared to be relatively strongly bound to proteins and is hardly attacked by uricase. After being free, it absorbs at shorter W.Ls, 283–285 nm (experiment [2]).

This might be understood on the basis of the old suggestion of gudzent [2] that both the lactam (keto) and the lactim (enol) toutomers of uric acid might be stable enough to coexist in biological fluids under certain circumstances.

In serum, these forms are expected to co-ordinate with proteins through hydrogen bonding. The energy of the hydrogen bond varies from 8.37 kJ for the H-N...H to 29.29 kJ for the H-O...H [3]. Therefore, it appears that the lactim toutomer will be relatively more strongly bound to proteins. Since the hydrogen bond by itself is a weak bond, this can explain why the loosely associated urate (lactam) is easily liberated as soon as the folded (quaternary) structure of proteins was disturbed. However the existence of protein-bound urate (PBU) could not be simply explained on the basis of hydrogen bond formation between the lactim toutomer of urate and proteins. Other type of co-ordinations might be involve to give that relatively stable protein-bound substance (PBU).

On the other hand, the hydrogen bond formation causes a relatively large amount of polarity in serum urate as compared to the pure uric acid solution. This may explain the spectral shifts in the absorbance maximum (283–290 nm) of serum urate compared to 293, 295 nm for the pure uric acid solution at the same pH (8.5, 9.35). Also this may explain the relative shift in the absorbance of the more polar lactim (PBU) to shorter W.Ls as compared to the relatively less polar lactam.

In addition, further investigation of the absorption spectra of serum between 270 nm and 300 nm before and after destruction with uricase demonstrate the presence of protein-bound substances which give their absorption spectra, after liberation from proteins, in the UV light at the W.Ls characteristic to uric acid or urate in serum and not affected by uricase. The rates of liberation of these substances differ than the rate of destruction of uric acid by uricase.

This represents the major obstacle against any attempt to record the real decrease in absorbance at 283–285 nm when serum proteins or the serum were subjected for the action of uricase. Actually, the results obtained are the difference between decrease in uric acid due to the destructive action of uricase and the increase of these liberated substances from proteins .

At the beginning, the rate of destruction of uric acid is higher than the rate of liberation of these substances, the result is a decrease in absorbance at 283–285 nm, the W.L. characteristic for urate max. absorbance in serum.

By the time the rate of liberation of these substances is increased in relation to the destruction rate of urate, this gives rise to increase in absorbance not only at the specific W.L characteristic to urate max. absorbance in serum, but also in the region from 270–300 nm.

Obviously, these substances will interfere with determination of serum urate if the measurements were carried out in the ultraviolet light after destruction with uricase.

At shorter U.V W.Ls,the blank is too high to record any change.