Plasma AcSDKP Levels Research Articles

Abstract MP43: Knockout Of ACE-N Facilitates Improved Cardiac Function After Myocardial Infarction

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal domain (ACE-N). We reported that cardioprotective effects of ACE inhibitor in angiotensin II-induced hypertension were partially mediated through increased Ac-SDKP bioavailability. Whether increased endogenous Ac-SDKP by knocking out ACE-N leads to improved cardiac function in myocardial infarction (MI) is unknown. Wild-type (WT) and ACE-N knockout (ACE-N -/- ) mice were subjected to MI induced by ligating the left anterior descending artery and treated with either vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks. Echocardiography was performed on awake mice at the end of experiment and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-N-/- mice showed increased plasma Ac-SDKP level in Sham or MI group compared to WT. Exogenous Ac-SDKP further increased Ac-SDKP level in both WT and ACE-N-/-. SF and EF were significantly decreased in both WT and ACE-N-/- mice post-MI, . Exogenous Ac-SDKP further increased EF and SF post-MI only in WT, but not in ACE-N-/- mice. Sarcoendoplasmic reticulum calcium transport ATPase 2 (SERCA2), a marker of cardiac calcium homeostatsis, significantly decreased in WT post-MI was rescued with Ac-SDKP, whereas ACE-N-/- mice displayed less reduction in SERCA2 expression. These results demonstrate that gene deletion of ACE-N improves cardiac function in mice post-MI, which was associated with increased Ac-SDKP level and minimally reduced expression of SERCA2.Therefore,this study illustrates that endogenous Ac-SDKP results in cardioprotective role in MI.

Dose-effect relationship of perindopril 10, 14 and 20 mg assessed by urine and plasma AcSDKP levels in mildly sodium-depleted healthy volunteers

BackgroundWe describe a pharmacodynamic study of the dose-effect relationship of perindopril arginine at 10, 14, and 20mg with in vivo angiotensin-converting enzyme (ACE) activity, assessed by urine and plasma AcSDKP levels, as well as the effect on plasma active renin concentrations and blood pressure. MethodsThis randomized, double-blind, four-period, crossover study involved single-dose administration of perindopril arginine (10, 14, and 20mg or placebo) to 32 healthy male normotensive mildly sodium-depleted volunteers. Blood and urine were collected over 48h for AcSDKP, ACE activity, and plasma active renin measurements. ResultsThere were dose-related increases in urinary AcSDKP excretion and plasma AcSDKP concentration after administration of perindopril, with significant between-period differences (estimate of the median difference in urinary excretion over 48h of AcSDKP, 49.1 [95% CI: 15.3–82.0] nmol for 14 versus 10mg, and 73.2 [95% CI: 44.9–106.3] nmol for 20 versus 14mg). Consequently, a dose-dependent increase in plasma active renin concentration was observed. Even though each dose of perindopril 10 to 20mg was associated with a significant 24-h ambulatory blood pressure reduction versus placebo, no dose-dependency was detected in these normotensive subjects. ConclusionsAdministration of perindopril arginine 10, 14, or 20mg to mildly sodium-depleted healthy volunteers is associated with a dose-dependent inhibition of in vivo ACE activity with significant between-dose differences. This effect was associated with a dose-dependent increase in plasma active renin concentration, indicating a dose-dependent blockade of the renin angiotensin system.

Clinical Implications of Plasma N-acetyl-seryl-aspartyl-lysyl-proline Level in Stable Kidney Transplant Recipients.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is partially eliminated in urine, accumulation of AcSDKP due to chronic renal failure may cause anemia. However, the status of plasma AcSDKP level in stable kidney transplant recipients is unknown although some recipients develop anemia after kidney transplantation. In this study, we investigated the relationship between plasma AcSDKP-like immunoreactive substance (IS) level and clinical characteristics associated with renal anemia in stable kidney transplant recipients. Forty Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma AcSDKP-IS levels were measured using an enzyme immunoassay. A significant correlation was observed between plasma AcSDKP-IS level and creatinine clearance. On the other hand, no significant correlation was observed between plasma AcSDKP-IS level and prolyl oligopeptidase activity, angiotensin II, or erythropoietin level. A significant difference in plasma AcSDKP-IS level was observed between recipients with no renal anemia and those with renal anemia. These results suggest that plasma AcSDKP level may depend largely on renal function and suggest a possibility that accumulation of AcSDKP may be partially involved in the pathogenesis of renal anemia in stable kidney transplant recipients.

Darbepoetin Alfa Increases Plasma N-acetyl seryl-aspartyl-lysyl-proline Level in Kidney Transplant Recipient: A Case Report

We report a case of elevated plasma N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP)-like immunoreactive substance (IS) level after multiple doses of a recombinant human erythropoietin (rHuEPO), darbepoetin alfa, in a 50 year-old Japanese female who underwent kidney transplantation. Plasma AcSDKP-IS levels showed a gradual decline after kidney transplantation. However, plasma AcSDKP-IS level showed a rapid rise on day 70, and remained at high levels for a period of time and then declined sharply on day 119. After maintaining relatively constant levels from day 119, plasma AcSDKP level showed a rapid rise again on day 168, and declined again on day 238. The two abrupt increases and subsequent sharp decreases of plasma AcSDKP-IS levels correlated temporally with the initiation and discontinuation of the two course of darbepoetin alfa. Although further detailed studies are necessary, administration of darbepoetin alfa may cause a marked increase in plasma AcSDKP level.

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N-terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP-like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β-D-galactosidase-labeled Gly-γAbu-SDKP as a marker antigen, an anti-rabbit IgG-coated immunoplate as a bound/free separator and 4-methylumbelliferyl-β-D-galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP-IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter-assay and 13.3 and 7.8% for intra-assay comparisons. Plasma AcSDKP-IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients.

On‐line solid‐phase extraction LC‐MS/MS for the determination of Ac‐SDKP peptide in human plasma from hemodialysis patients

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-β fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.

Quantification of N-acetyl-seryl-aspartyl-lysyl-proline in hemodialysis patients administered angiotensin-converting enzyme inhibitors by stable isotope dilution liquid chromatography–tandem mass spectrometry

We developed a sensitive, selective and accurate method based on liquid chromatography with tandem mass spectrometry (LC–MS/MS) to determine N-terminal thymosin-β peptides of Ac-SDKP and Ac-ADKP in human plasma samples. Quantification of Ac-SDKP and Ac-ADKP was performed using solid phase extraction (SPE) based on C18, reversed phase LC separation, and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-13C6, 15N2 and Ac-ADKP-d7 were synthesized for the internal standards. These MRM monitoring ions were m/z 488→129 (quantitative ion)/226 for Ac-SDKP, m/z 496→137 for Ac-SDKP-13C6, 15N2, m/z 472→129 (quantitative ion)/226 for Ac-ADKP, and m/z 479→129 for Ac-ADKP-d7, respectively. Lower limit of quantitation (LLOQ) of Ac-SDKP and Ac-ADKP was 0.1ng/mL in human plasma. Recovery values were ranged from 94.7% to 106.3% for inter- (RSD: 0.6–3.5%) and intra- (RSD: 0.4–4.9%) day assays. Plasma Ac-SDKP levels were significantly higher in hemodialyzed subjects treated with angiotensin-converting enzyme inhibitors of enalapril (27.3±24.6ng/mL, n=10) and trandolapril (12.3±16.9ng/mL, n=18) than healthy (0.4±0.2ng/mL, n=7) and hemodialyzed subjects (0.6±0.2ng/mL, n=34). This analytical method would be useful to measure N-terminal thymosin-β peptides in human plasma for the clinical study.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Inhibits TGF-β–Mediated Plasminogen Activator Inhibitor-1 Expression via Inhibition of Smad Pathway in Human Mesangial Cells

Recent large clinical trials indicate that angiotensin-converting enzyme inhibitors (ACE-I) attenuate the detrimental outcome of progressive renal disease. The hemoregulatory tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP, AcSDKP) is hydrolyzed by ACE, and plasma Ac-SDKP level is increased by fivefold after treatment with ACE-I. Ac-SDKP was found to ameliorate cardiac and renal fibrosis in hypertensive animal models. However, the molecular mechanisms by which Ac-SDKP mediates anti-fibrotic effects remain unclear. This study is an examination of the interaction between Ac-SDKP and transforming growth factor-beta (TGF-beta), one of the key cytokines in the progression of renal disease, in human mesangial cells. Ac-SDKP inhibited TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1) and alpha2 (I) collagen mRNA. Ac-SDKP suppressed not only TGF-beta1-induced Smad2 phosphorylation at Ser-465/467 in a dose-dependent manner, but also the nuclear accumulation of receptor-regulated Smads (R-Smad), Smad2 and Smad3. As expected, Ac-SDKP inhibited TGF-beta-responsive Smad-dependent luciferase reporters, 3TP-luc and 4xSBE-luc. Immunofluorescence analysis revealed that the inhibitory Smad, Smad7, was exported to the cytoplasm from the nucleus by the treatment with Ac-SDKP. These findings provide novel evidence that Ac-SDKP inhibits TGF-beta signal transduction through the suppression of R-Smad activation via nuclear export of Smad7, highlighting an alternative mechanism involved in the reno-protective efficacy of ACE-I.

Angiotensin I-converting enzyme and metabolism of the haematological peptide N-acetyl-seryl-aspartyl-lysyl-proline.

1. Angiotensin I-converting enzyme (ACE) has two homologous active N- and C-terminal domains and displays activity towards a broad range of substrates. The tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) has been shown to be hydrolysed in vitro by ACE and to be a preferential substrate for its N-terminal active site. This peptide reversibly prevents the recruitment of pluripotent haematopoietic stem cells and normal early progenitors into the S-phase. 2. Angiotensin I-converting enzyme inhibitors, given as a single dose to normal subjects or during long-term treatment in hypertensive patients, result in plasma AcSDKP levels five- to six-fold higher and urine concentrations 40-fold higher than those of control subjects and/or patients. Thus, AcSDKP is a natural peptide hydrolysed by the N-terminal domain of ACE in vivo. In addition, ACE may be implicated in the process of haematopoietic stem cell regulation by permanently degrading this natural circulating inhibitor of cell entry into the S-phase. 3. Besides hydrolysis by ACE, the second very effective mechanism by which AcSDKP is cleared from plasma is glomerular filtration. Because of its high sensitivity and specificity, the measurement of AcSDKP in plasma and urine provides a valuable tool in screening specific inhibitors of the N-terminal domain of ACE and in monitoring ACE inhibition during chronic treatment. 4. The long-term consequences of AcSDKP accumulation are not known. During chronic ACE inhibition in rats, AcSDKP levels slightly increase in organs with high ACE content (kidneys, lungs). To significantly increase its concentration in target haematopoietic organs (the extracellular fraction of bone marrow), AcSDKP has to be infused on top of a captopril-based treatment. 5. A selective inhibitor of the N-domain of ACE in vitro and in vivo has been identified recently. The phosphinic peptide RXP 407 does not interfere with blood pressure regulation, but does increase, dose dependently, plasma concentrations of AcSDKP in mice, in contrast with lisinopril, which affects the metabolism of both AcSDKP and angiotensin I. N-Terminal-selective ACE inhibitors may be used to selectively control AcSDKP metabolism in target haematopoietic organs. This new therapeutic strategy may be of value for protecting haematopoietic cells from the toxicity of cancer chemotherapy.

Chronopharmacologic aspects of continuous AcSDKP infusion in mice

Inconsistent results characterized N-acetyl-Ser-Asp-Lys-Pro (AcSDKP or Goralatide) effects upon hematologic proliferation, possibly because its circadian organization had been overlooked. We investigated the circadian changes in AcSDKP disposition in plasma and in bone marrow during continuous infusion and AcSDKP effects upon the circadian rhythms in bone marrow granulomonocytic precursors (CFU-GM) and circulating blood cell counts. One hundred ninety-six male B6D2F1 mice received a constant infusion of AcSDKP (24 μg/day) or 0.9% NaCl for 7 days, using an osmotic minipump. All mice were synchronized with an alternation of 12 hours of light and 12 hours of darkness for 3 weeks prior to study. Mice were sacrificed on the fifth or seventh infusional day at 3, 9, 15, or 21 hours after light onset (HALO) in order to assess plasma and bone marrow AcSDKP concentrations, CFU-GM, and/or circulating blood cell counts. In control mice, plasma and bone marrow AcSDKP concentrations displayed a circadian rhythm with a maximum level during the dark span, at 21 and 15 HALO respectively, while CFU-GM, leukocyte, lymphocyte, and monocyte counts peaked during early light. Continuous AcSDKP infusion increased fivefold mean plasma AcSDKP level at 3 or 9 HALO, thus inverted its physiologic rhythm and suppressed the CFU-GM peak that normally occurs at these times. This inhibition however, was indirect, because the rhythms in bone marrow AcSDKP concentration were similar with or without AcSDKP infusion. Conversely, mean leukocyte and lymphocyte counts were significantly reduced with AcSDKP infusion, while their circadian rhythms remained unaffected and were amplified. The results indicate that AcSDKP pharmacology displays circadian rhythmicity and warrant the exploration of chronopharmacologic schedules of AcSDKP delivery for further protecting bone marrow against chemotherapy insults.

High plasma level of N-acetyl-seryl-aspartyl-lysyl-proline: a new marker of chronic angiotensin-converting enzyme inhibition.

The acute administration of the angiotensin-converting enzyme (ACE) inhibitor captopril to healthy subjects transiently increases 5.5-fold the plasma levels of a natural stem-cell regulator, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). The aim of this study was to measure plasma Ac-SDKP levels during chronic treatment with all types of ACE inhibitors and to assess its relevance as a marker of ACE inhibition. Plasma levels of Ac-SDKP were blindly determined in age- and sex-matched hypertensive patients either treated (ACEI group, n=27) or not (non-ACEI group, n=23) with an ACE inhibitor for more than 1 month. Geometric mean [range] of plasma Ac-SDKP levels were significantly higher in the ACEI group (3.78 [1.48 to 14.5] pmol/mL) than in the non-ACEI group, with no overlap between the groups (0.75 [0.36 to 1.22] pmol/mL, P<.0001). The measurement of Ac-SDKP in plasma discriminated all the patients of the ACEI group, whereas the simultaneous determination of either in vitro (using hippuryl-histidine-leucine as substrate) or in vivo (angiotensin II/angiotensin I ratio) ACE activity failed to identify nine and five cases, respectively. We conclude that Ac-SDKP accumulates in plasma during chronic ACE inhibitor treatment. The long-term consequences of Ac-SDKP accumulation are unknown. The reliability of plasma Ac-SDKP measurement makes it the best marker of chronic ACE inhibition, which can help to verify patients' compliance to ACE inhibitor treatment.

Effects of the angiotensin-converting enzyme inhibitor enalapril on blood haematopoietic progenitors and acetyl-N-Ser-Asp-Lys-Pro concentrations.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) is a physiological inhibitor of the proliferation of haematopoietic stem cells. In 12 healthy volunteers treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril (20 mg day-1 for 15 days), we studied plasma and urinary AcSDKP levels, the in vitro degradation of AcSDKP by plasma ACE and the numbers of circulating haematopoietic progenitors (granulocyte-monocytic colony forming unit: CFU-GM; burst forming unit-erythroid: BFU-E; and mixed colony forming unit: CFU-mixed). During treatment, plasma and urinary AcSDKP concentrations increased 2- to 5-fold, degradation of AcSDKP was reduced, and CFU-mixed significantly increased by 100% while BFU-E and CFU-GM significantly decreased by 16% and 26%, respectively. These results indicate that ACE inhibitors may be of value during chemotherapy or radiotherapy, warranting further study.

Substrate Dependence of Angiotensin I-Converting Enzyme Inhibition: Captopril Displays a Partial Selectivity for Inhibition ofN-Acetyl-Seryl-Aspartyl-Lysyl-Proline Hydrolysis Compared with That of Angiotensin I·

Angiotensin I-converting enzyme (ACE) is composed of two highly similar domains (referred to here as the N and C domains) that play a central role in blood pressure regulation; ACE inhibitors are widely used in the treatment of hypertension. However, the negative regulator of hematopoiesis, N-acetyl-seryl-aspartyl-lysyl-prolyl (AcSDKP), is a specific substrate of the N domain-active site; thus, in addition to the cardiovascular function of ACE, the enzyme may be involved in hematopoietic stem cell regulation, raising the interest of designing N domain-specific ACE inhibitors. We analyzed the inhibition of angiotensin I and AcSDKP hydrolysis as well as that of three synthetic ACE substrates by wild-type ACE and the N and C domains by using a range of specific ACE inhibitors. We demonstrate that captopril, lisinopril, and fosinoprilat are potent inhibitors of AcSDKP hydrolysis by wild-type ACE, with K(i) values in the subnanomolar range. However, of the inhibitors tested, captopril is the only compound able to differentiate to some degree between AcSDKP and angiotensin I inhibition of hydrolysis by wild-type ACE: the K(i) value with AcSDKP as substrate was 16-fold lower than that with angiotensin I as substrate. This raises the possibility of using captopril to enhance plasma AcSDKP levels with the aim of normal hematopoeitic stem cell protection during chemotherapy and a limited effect on the cardiovascular function of ACE.