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Neuropeptide Y (NPY) is a 36-amino acid peptide circulating at a subpicomolar concentration participating in multiple physiological and pathological processes. NPY is prone to peptidolysis, generating metabolites with modified affinity for the five known receptors of NPY that mediate distinct effects. It is, therefore, crucial to distinguish each metabolite to understand the multiple functions of NPY. Since immunoassays are not able to distinguish NPY from its metabolites, we have validated a microliquid chromatography tandem mass spectrometry (micro-LC-MS/MS) assay for the quantification of endogenous NPY, NPY2–36, NPY3–36, NPY1–35, and NPY3–35 in human plasma. Sample preparation relies on immunoextraction in 96-well plates, followed by solid-phase extraction prior to micro-LC-MS/MS. The LLOQ ranged from 0.03 to 0.16 pM, intra- and inter-assay precision were <27% and trueness <22%. We determined reference intervals in 155 healthy volunteers and 40 hypertensive patients. We found that NPY3–36 is the main circulating peptide in resting conditions and that NPY and catecholamines are simultaneously increased during orthostasis. We also showed that the concentrations of NPY and its metabolites are similar in healthy volunteers and hypertensive patients. NPY is the prototype peptide that circulates in concentrations expected to be beyond instrumental capacities. We have been successful in developing a high-throughput specific and sensitive assay by including a deep knowledge of the physicochemical properties of these peptides to an efficient multistep sample preparation, and a micro-LC chromatography. We believe that our methodological approach opens the possibility to selectively quantify other endogenous peptides cleaved by peptidases whose concentrations are below 1 pM.

Introduction:Preclinical systolic dysfunction (PSD), also classified as ACC/AHA Stage B heart failure, is associated with increased cardiac morbidity. Impaired renal response is a hallmark of PSD with decreased renal cyclic guanosine monophosphate (cGMP) generation to acute volume load (AVL). We tested the hypothesis that chronic phosphodiesterase V (PDEV) inhibition would improve renal function and potentiate urinary sodium and cGMP excretion in subjects with PSD in response to AVL.Methods:Double-blinded placebo-controlled study in 20 PSD subjects who were randomized in a 2:1 fashion to receive tadalafil 20 mg daily (n=14) or placebo (n=6). Subjects underwent two study days, at baseline and after 12 weeks of study drug. On each study day, an echocardiogram, renal clearance study and humoral assessment were obtained before and after acute volume expansion (normal saline 0.25 ml/kg/min for 1 hour). Changes were summarized with median and quartiles and groups were compared using two-sample t-test.Results:Baseline characteristics were similar between groups. We confirmed that there was no increase in urinary sodium and cGMP excretion in response to AVL at baseline. At 12 weeks, tadalafil reduced LA volume index (-2.5 (-8,2) vs 5 (3,5) ml/m2, p=0.04) without a significant change in LV EF, plasma cGMP and urinary cGMP as compared to placebo. Treatment with tadalafil resulted in increased cardiac index (0.3 (0.3,0.5) vs -0.15 (-0.4, 0.01) L/m2, p=0.01), however it did not improve renal function, urinary sodium or cGMP excretion as compared to placebo in response to AVL.Conclusion:Chronic PDEV inhibition (12 weeks) with tadalafil in PSD resulted in reduction of LAVI and improved cardiac index in response to AVL without improvement in renal response. Impaired handling of sodium load may contribute to the progression of HF, hence the current study suggests that PDEV inhibition may not be useful in preventing progression of PSD to clinical HF.