Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 49

significantly decreased for Blmh / mice compared with Blmh +/+ animals (33.1 min
vs. 41.2 min, P ¼ 0.012). While only one mouse out of 44 Blmh +/+ mice (2.3 %)

dies (at 61 min) after L -Hcy-thiolactone injection, the incidence of death is signifi-
cantly increased for Blmh / mice (to 46.9 %, P <

0.001) (Table 3.10 ) [141].
Although in the i.p. injection experimental model Hcy is also generated, Hcy levels
are decreased by the inactivation of the Blmh gene. Thus, in this model, neurotox-
icity can be assigned to Hcy-thiolactone, but not to Hcy. Taken together, the
experiments with Blmh-null mouse model provide direct evidence that Hcy-
thiolactone, rather than Hcy itself, is neurotoxic in vivo [141].

3.5.2 Urinary Excretion

In humans and mice endogenous Hcy-thiolactone is also eliminated by urinary
excretion [93, 95]. Hcy-thiolactone concentrations in urine vary from 11 nM to
485 nM and are 100-fold higher than in plasma. Urinary Hcy-thiolactone accounts
for 2.5-28 % of urinary tHcy. Relative renal clearance of Hcy-thiolactone is 0.2-7.0
of creatinine clearance, while clearance of tHcy is only about 0.001-0.003 [95].
Efficient urinary elimination of Hcy-thiolactone is typical for the waste or toxic
products of normal human metabolism.
Calculations based on a normal human glomerular filtration rate of 180 L/day
and a free plasma Hcy concentrations of 3

M indicate that 99 % of filtered tHcy is
reabsorbed [289]. A similar calculation for Hcy-thiolactone (0.12-2.4 nM in plasma
and 286-415 nmol/day eliminated with urine) indicates that only 0.4-3.8 % is
reabsorbed and

μ

95 % of filtered Hcy-thiolactone was excreted in humans [95].
Urinary Hcy-thiolactone levels are negatively correlated to urinary pH
(Fig. 3.9a ). In contrast, urinary pH is not correlated to urinary tHcy levels
(Fig. 3.9b ). A possible mechanism facilitating the accumulation of Hcy-thiolactone
in urine and explaining the pH dependence of urinary elimination of Hcy-
thiolactone involves a gain of positive charge by Hcy thiolactone, which prevents
its reabsorption by the renal tubules. Hcy-thiolactone has a pK a ¼ 6.67 [84] and
exists in the positively charged acid form and the neutral base form (Reaction 3.3 ).
Thus, at pH 7.4 in the blood, Hcy-thiolactone exists in the mostly neutral base form,
whereas at pH 5-6 in the urine, the positively charged form predominates. Urinary
acidification apparently maintains low fractional concentration of the uncharged
base form of Hcy-thiolactone inside tubular lumen. This sustains continuous Hcy-
thiolactone diffusion from the tubular cells (with high fractional concentration of
the base form) into the lumen (with low fractional concentration of the base form).
In mice fed a normal chow diet, urinary Hcy-thiolactone concentration is
140 nM [93], similar to urinary Hcy-thiolactone value in humans [95]. However,
in mice fed a hyperhomocysteinemic high-Met diet, urinary Hcy-thiolactone
increases 25-fold, compared to mice fed a normal diet. The distributions of Hcy-
thiolactone between plasma and urine in mice fed a normal diet and humans are

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