Packed Red Blood Cells Are an Abundant and Proximate Potential Source of Nitric Oxide Synthase Inhibition

<div><p>Objective</p><p>We determined, for packed red blood cells (PRBC) and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethylarginine (LNMMA).</p><p>Background</p><p>ADMA and LNMMA are near equipotent NOS inhibitors forming blood’s total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined.</p><p>Methods</p><p>We measured total (free and protein incorporated) ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete <i>in vitro</i> acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis.</p><p>Results</p><p><i>In vitro</i> strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM) and LNMMA (58.9 ± 28.9 μM) that persisted over 42-d at 6° or -80°C. <i>In vitro</i> 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma.</p><p>Conclusion</p><p>The compelling physiological ramifications are that regardless of storage age, 1) PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2) PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon transfusion.</p></div

Hematologic values for PRBCs in Catalase experiment.

<p>*The average value for μmol ADMA/ g catalase was determined separately on pure human catalase following strong acid hydrolysis. This value was multiplied by the measured catalase concentration in g/L PRBC to give the estimated μmol ADMA/L of PRBC.</p><p>Hematologic values for PRBCs in Catalase experiment.</p

Relative methylated arginine release over 5 h from PRBC incubation.

<p>All three methylated arginines, ADMA, LNMMA, and symmetric dimethylarginine were released in proportionately similar amounts during the 5h incubation at 37°C. The starting free concentrations were different (0.58 ± 0.12, 0.08 ± 0.02, and 0.20 ± 0.12 respectively). Free ADMA, LNMMA and symmetric dimethylarginine increased significantly from baseline by 5h. Linear regression analysis failed to detect differences among methylated arginines over 5h.</p

Arginine and its endogenous methylated derivatives.

<p>Arginine is the normal substrate for NOS resulting in NO formation. A single methylation of arginine produces monomethylarginine (LNMMA) which, along with asymmetric dimethylarginine (ADMA), are endogenous inhibitors of NOS. ADMA and LNMMA are hydrolyzed by dimethylarginine dimethylaminohydrolase (DDAH). Symmetric dimethylarginine does not inhibit NOS. These structures were drawn as they exist at mammalian physiological pH using ChemDraw software (PerkinElmer Informatics) and data from PubChem at NCBI at the National Library of Medicine (USA).</p

Incubation-induced release of Free ADMA over 5h by Blood Product type.

<p>Incubation of defrosted fresh frozen plasma (open triangles), supernatants of PRBC alone (solid squares), a mixture of 1:1 PRBC and fresh frozen plasma (open circles), and a mixture of 1:3 PRBC and fresh frozen plasma (open diamonds). Repeated measures Two-way ANOVA shows significant differences of ADMA among PRBC and PRBC: fresh frozen plasma groups over the incubation period (p<0.006). Tukey’s <i>post-hoc</i> multiple comparison test revealed significant differences of ADMA (α = 0.05) among groups at individual sampling points. At three and five hour time points, ADMA in PRBC and 1:1 PRBC: fresh frozen plasma was significantly (*) higher than 1:3 PRBC: fresh frozen plasma. Hemoglobin measurements confirmed PRBC concentration and dilutions.</p

Total methylarginines obtained by strong acid hydrolysis of pre-aliquoted PRBCs stored at 6°C or -80°C.

<p>No statistically significant differences were detected between 6°C or -80°C storage at any time (pooled to 5, 14, 28 and 42 d) for ADMA, LNMMA and symmetric dimethylarginine and no change from control was detected. It is likely that neither PRMT activity nor DDAH-induced hydrolysis was a dominating effect under these storage conditions. However, equal formation and hydrolysis cannot be ruled out.</p

ADMA scaled against hemoglobin concentration over storage time at 6°C or -80°C.

<p>In an attempt to account for any dehydration of the samples under either storage condition (6°C or -80°C), we measured hemoglobin concentrations paired with each ADMA measurement to calculate ADMA to hemoglobin ratio. The time course and pattern remained unchanged over time suggesting no measurable desiccation effect on sample concentration over the 42-day storage.</p