Pre-Analytical and Clinical Validation of a Dried Blood Spot Assay for Asymmetric Dimethylarginine and L-Arginine

Figures

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  Figure 1. Effect of air humidity on dried blood spot assay results. Concentrations of asymmetric dimethylarginine (ADMA) (a) and L-arginine (b) in untreated dried blood spots subjected to low (40–60%) or high air humidity (95%), as compared to dried blood spots pre-treated with the arginase inhibitor, nor-NOHA, at high air humidity. Data are presented as mean ± SD; * p < 0.05 between groups.

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  Figure 2. Combined effect of temperature and humidity on dried blood spot assay results. Filter cards were exposed to 20 °C or 35 °C, either at low (40–60%) or high air humidity (95%) for one month, and ADMA (a) and L-arginine (b) were measured from the dried blood spots. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01 between groups; n.s.: not significant.

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  Figure 3. Effect of long-term storage at room temperature on dried blood spot assay results. ADMA and L-arginine were measured from dried blood spots on filter cards that had been pretreated with nor-NOHA and stored for up to 307 days at room temperature. Data are presented as mean ± SD; * p < 0.05 vs. day 0.

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  Figure 4. Correlation of ADMA and L-arginine concentrations measured in dried blood spots and in plasma. ADMA (a) and L-arginine (b) were measured from dried blood spots and plasma samples collected from the same subjects at the same day. Each dot marks one subject’s assay results. The dotted line marks the line of equivalence between both assays. R values represent Pearson’s correlation coefficients.

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  Figure 5. Box plots representing concentrations of ADMA and L-arginine at baseline and after 6 months of chronic intermittent hypoxia (CIH). ADMA (a) and L-arginine (b) were measured from dried blood spots and plasma in individuals exposed to six months of chronic intermittent hypoxia (CIH). * p <0.05 between groups.

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  Figure 6. Association of baseline ADMA concentration in dried blood spots with mean pulmonary arterial pressure after 6 months of CIH. (a) Linear regression analysis of dried blood spot ADMA concentration at baseline with mean pulmonary arterial pressure (mPAP) after six months of exposure to chronic intermittent hypoxia (CIH). (b) Mean pulmonary arterial pressure (mPAP) at month 6 of CIH exposure in individuals with baseline dried blood spot ADMA concentration below or above o.615 µmol/L, the optimal cut-off for discrimination of the two groups in ROC analysis. Horizontal lines indicate mean ± SD; each dot represents one individual. ** p < 0.01 between groups.

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  Figure 7. Clinical validation of dried blood spot ADMA and L-arginine. (a) ADMA concentration in dried blood spots in male and female subjects; (b) L-arginine concentration in dried blood spots in individuals taking L-arginine supplements or not; (c) L-arginine/ADMA ratio according to number of traditional risk factors present. Data are presented as mean ± SD; * p < 0.05 between groups in (a) and (b); * p < 0.05 for trend across risk factor groups in (c).

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  Figure 8. Distribution of ADMA concentrations (a) and L-arginine concentrations (b) in dried blood spots.