Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system.

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0-15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins.

Kallidin-like peptide mediates the cardioprotective effect of the ACE inhibitor captopril against ischaemic reperfusion injury of rat heart.

1. The potential cardioprotective effect of ACE inhibitors has been attributed to the inhibition of bradykinin degradation. Recent data in rats documented a kallidin-like peptide, which mimics the cardioprotective effect of ischaemic preconditioning. This study investigates in isolated Langendorff rat heart the effect of the ACE inhibitor captopril, the role of bradykinin, kallidin-like peptide, and nitric oxide (NO). 2. The bradykinin level in the effluent of the control group was 14.6 pg ml(-1) and was not affected by captopril in the presence or absence of kinin B2-receptor antagonist, HOE140. 3. The kallidin-like peptide levels were approximately six-fold higher (89.8 pg ml(-1)) and increased significantly by treatment with captopril (144 pg ml(-1)), and simultaneous treatment with captopril and HOE140 (197 pg ml(-1)). 4. Following 30 min ischaemia in the control group, the creatine kinase activity increased from 0.4 to 53.4 U l(-1). In the captopril group and in the captopril+L-NAME group, the creatine kinase activity was significantly lower (18.5 and 22.8 U l(-1)). This beneficial effect of captopril was completely abolished by the kinin B2-receptor antagonist, HOE140, as well as by the kallidin antiserum. 5. Perfusion of the hearts with kallidin before the 30 min ischaemia, but not with bradykinin, yielded an approximately 50% reduction in creatine kinase activity after reperfusion. 6. Pretreatment with L-NAME alone and simultaneously with captopril, and with kallidin, respectively, suggests a kinin-independent action of NO before the 30 min ischaemia on coronary flow and a kinin-dependent action after ischaemia. 7. These data show that captopril increases kallidin-like peptide in the effluent. Kallidin-like peptide via kinin B2 receptor seems to be the physiological mediator of cardioprotective actions of captopril against ischaemic reperfusion injury. HOE140 as well as the kallidin antiserum abolished the cardioprotective effects of captopril.

Losartan increases bradykinin levels in hypertensive humans.

BACKGROUND: Studies in animals and humans indicate a role for kinins in the actions of angiotensin type 1 (AT1) receptor blockers. However, the effect of these compounds on kinin levels in humans is unknown. METHODS AND RESULTS: We measured angiotensin (Ang), bradykinin (BK), and kallidin peptides in subjects with essential hypertension administered placebo, losartan (50 mg OD), and eprosartan (600 mg OD) in randomized order in a double-blind, 3-period, 3-treatment, crossover trial. Peptides were measured in arterial blood using high-performance liquid chromatography-based radioimmunoassays. Losartan increased blood levels of BK-(1-9) and hydroxylated BK-(1-9) by approximately 2-fold and reduced the BK-(1-7)/BK-(1-9) ratio by 55%. There was a trend for eprosartan to produce similar changes in bradykinin levels. There were no changes in blood kallidin levels. Both losartan and eprosartan increased plasma levels of Ang I, Ang II, and Ang-(2-8), and eprosartan increased Ang-(3-8) levels. Ang-(1-7) and Ang-(1-9) levels were unchanged. There was an associated 30% to 35% reduction in Ang II/Ang I ratio and 63% to 69% reduction in Ang-(1-7)/Ang I ratio. Plasma ACE activity was unchanged. CONCLUSIONS: Losartan increases bradykinin levels. The reductions in BK-(1-7)/BK-(1-9), Ang II/Ang I, and Ang-(1-7)/Ang I ratios suggest that the increased bradykinin levels were the result of reduced metabolism by ACE and neutral endopeptidase. Increased bradykinin levels may represent a class effect of AT1 receptor blockers that contributes to their therapeutic actions and may also contribute to the angioedema that may accompany this therapy.

Activation of the tissue kallikrein-kinin system in stroke.

Edema formation is a major problem in large ischemic infarcts, and the underlying breakdown of the blood-brain barrier is only incompletely understood. Here, we report that the tissue kallikrein-kinin system, which influences the permeability of the blood-brain barrier, is activated in stroke. In 22 patients with large infarcts in the territory of the middle cerebral artery, we found elevated plasma concentrations of the tissue kinin kallidin. The data suggest that further studies on a possible role of kinin receptor antagonists on edema after stroke are warranted.

Activation of the kallikrein-kinin system by cardiopulmonary bypass in humans.

We used cardiopulmonary bypass (CPB) as a model of activation of the contact system and investigated the involvement of the plasma and tissue kallikrein-kinin systems (KKS) in this process. Circulating levels of bradykinin and kallidin and their metabolites, plasma and tissue kallikrein, low and high molecular weight kininogen, and kallistatin were measured before, during, and 1, 4, and 10 h after CPB in subjects undergoing cardiac surgery. Bradykinin peptide levels increased 10- to 20-fold during the first 10 min, returned toward basal levels by 70 min of CPB, and remained 1.2- to 2.5-fold elevated after CPB. Kallidin peptide levels showed little change during CPB, but they were elevated 1.7- to 5.2-fold after CPB. There were reductions of 80 and 60% in plasma and tissue kallikrein levels, respectively, during the first minute of CPB. Kininogen and kallistatin levels were unchanged. Angiotensin-converting enzyme inhibition did not amplify the increase in bradykinin levels during CPB. Aprotinin administration prevented activation of the KKS. The changes in circulating kinin and kallikrein levels indicate activation of both the plasma and tissue KKS during activation of the contact system by CPB.

Kinins in humans.

The kinin peptide system in humans is complex. Whereas plasma kallikrein generates bradykinin peptides, glandular kallikrein generates kallidin peptides. Moreover, a proportion of kinin peptides is hydroxylated on proline(3) of the bradykinin sequence. We established HPLC-based radioimmunoassays for nonhydroxylated and hydroxylated bradykinin and kallidin peptides and their metabolites in blood and urine. Both nonhydroxylated and hydroxylated bradykinin and kallidin peptides were identified in human blood and urine, although the levels in blood were often below the assay detection limit. Whereas kallidin peptides were more abundant than bradykinin peptides in urine, bradykinin peptides were more abundant in blood. Bradykinin and kallidin peptide levels were higher in venous than arterial blood. Angiotensin-converting enzyme inhibition increased blood levels of bradykinin, but not kallidin, peptides. Reactive hyperemia had no effect on antecubital venous levels of bradykinin or kallidin peptide levels. These studies demonstrate differential regulation of the bradykinin and kallidin peptide systems, and indicate that blood levels of bradykinin peptides are more responsive to angiotensin-converting enzyme inhibition than blood levels of kallidin peptides.

Low-salt diet downregulates plasma but not tissue kallikrein-kinin system.

The kallikrein-kinin system (KKS) is involved in the regulation of blood pressure and in the sodium and water excretion. In humans, the KKS is divided functionally into a plasma KKS (pKKS) generating the biologically active peptide bradykinin and into the tissue (glandular) KKS (tKKS) generating the active peptide kallidin. The objective of this study was to examine the effect of a low-NaCl diet on the concentration of both pKKS and tKKS in plasma and urine in 10 healthy volunteers. After a 4-day low-NaCl diet, the urinary sodium and chloride excretions had decreased from 234 to 21.2 mmol/24 h and from 198 to 14.6 mmol/24 h, respectively. The plasma levels of ANG I, aldosterone, and angiotensin converting enzyme (ACE) significantly increased from 50.4 to 82.8 pg/ml, from 129 to 315 pg/ml, and from 46.4 to 59.8 U/ml, respectively, demonstrating the physiological adjustment to the low-salt diet. In plasma, the levels of bradykinin and plasma kallikrein had significantly decreased from 13.7 to 7.57 pg/ml and 14.4 to 7.13 U/ml, respectively. However, the levels of high-molecular-weight kininogen (HMW kininogen) remain unchanged (101 vs. 112 microg/ml, not significant). Contrary to plasma kallikrein, the plasma levels of tissue kallikrein increased (0.345 vs. 0.500 U/ml; P < 0.01). The plasma kallidin levels, however, did not change (64.7 vs. 68.6 pg/ml, not significant). This can be explained by a simultaneous decrease in the plasma low-molecular-weight kininogen (LMW kininogen) levels (89.9 vs. 44.4 microg/ml; P < 0.05). As in plasma, we find increased urinary concentrations of renal (tissue) kallikrein (23.3 to 42.8 U/24 h; P < 0.05) that contrast with, and are presumably counterbalanced by, urinary LMW kininogen levels (77.0 vs. 51.8 microg/24 h; P < 0.05). Consequently, in urine low-NaCl diet caused no significant change in either bradykinin or kallidin (9.2 vs. 10.8 microg/24 h, and 10.9 vs. 10.3 microg/24 h). It is concluded that the stimulation of the renin-angiotensin system on a low-NaCl diet is associated with a decrease in pKKS (bradykinin and plasma kallikrein) but not in tissue and renal KKS. Although tissue kallikrein is increased, there is no change in kallidin, as LMW kininogen in plasma and urine is decreased. These data suggest a difference in the regulation of pKKS and tKKS by low-salt diet.

Strategy of measuring bradykinin and kallidin and their concentration in plasma and urine.

Bradykinin (BK) and kallidin (KAL) derivatives containing a Cys residue instead of a Ser residue at positions 6 and 7, respectively [BK(Cys6), KAL(Cys7)], were synthesized. These derivatives were linked to BSA via the Cys residue by a heterobifunctional cross-linker. The coupling product containing a kinin with both free N- and C-terminal ends was used as immunogen. We obtained highly sensitive and specific antisera, simultaneously directed against both free ends. The radioimmunoassay for BK displays a sensitivity of 0.5-60 fmol BK at a dilution of 1:80,000 with 125I-BK(Tyr8) as tracer. Des-Arg9-BK, [BK(1-8)], displayed the highest cross-reactivity in the amount of 24%. Des-Arg1-BK and smaller molecular weight fragments display a cross-reactivity of less than 0.1%. The cross-reactivity of the BK antiserum with KAL is approximately 4%. In presence of 125I-KAL(Tyr9) the radioimmunoassay for KAL displays a sensitivity of 2 to 200 fmol KAL to an antiserum dilution of 1:80,000. The cross-reactivity with BK is 0.02%. KAL(Hyp4), BK(Hyp3), and des-Arg10-KAL [KAL(1-9)] show a cross-reactivity of 6.3, 4.9, and 2.4%. All other natural kinin derivatives show a cross-reactivity of less than 1%. Both assays were used to measure BK and KAL concentrations in blood and urine in humans after extraction and HPLC separation. The BK plasma level 1.97 (SD 0.54) pg/ml. The KAL plasma level is 81.0 (SD 14.3) pg/ml, indicating that KAL instead of BK is a circulating peptide. In urine, the BK level is 16.3 pg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of a vasoactive peptide related to lysyl-bradykinin from trout plasma.

Incubation of plasma from the steelhead trout, Oncorhynchus mykiss with porcine pancreatic glandular kallikrein generated bradykinin-like immunoreactivity. The primary structure of the immunoreactive peptide was established as: Lys-Arg-Pro-Pro-Gly-Trp-Ser-Pro-Leu-Arg. This sequence shows two amino acid substitutions (Phe6-->Trp and Phe9-->Leu) compared with mammalian lysyl-bradykinin (kallidin). Bolus intraarterial injection of the purified peptide produced a strong and sustained vasopressor response in the unanaesthetized trout. The data demonstrate that the kallikrein-kinin system predates the appearance of tetrapods and suggest a role for this system in cardiovascular regulation in fish.

Metabolism of bradykinin agonists and antagonists by plasma aminopeptidase P.

In addition to angiotensin I converting enzyme (ACE; EC 3.4.15.1) and carboxypeptidase N (CPN; EC 3.4.17.3), other peptidases contribute to bradykinin (BK) degradation in plasma. Rat plasma degraded BK by hydrolysis of the N-terminal Arg1-Pro2 bond, and the characteristics of hydrolysis are consistent with identification of aminopeptidase P (APP; EC 3.4.11.9) as the responsible enzyme. BK and BK[1-5] N-terminal hydrolysis was optimal at neutral pH, was inhibited by 2-mercaptoethanol, dithiothreitol, o-phenanthroline and EDTA, but was unaffected by the aminopeptidase inhibitors amastatin, puromycin and diprotin A, the endopeptidase-24.11 inhibitors phosphoramidon and ZINCOV, and the ACE and CPN inhibitors captopril and D,L-mercapto-methyl-3-guanidinoethylthiopropanoic acid (MERGETPA), respectively. Although kallidin (Lys-BK) was not metabolized directly by APP, conversion to BK by plasma aminopeptidase M (EC 3.4.11.2) resulted in subsequent degradation by APP. BK analogs containing N-terminal Arg1-Pro2 bonds, including [Tyr8-(OMe)] BK and [Phe8 psi(CH2NH)Arg9]BK (B2 agonists), des-Arg9-BK and [D-Phe8]des-Arg9-BK (B1 agonists), and [Leu8]des-Arg9-BK (B1 antagonist), were degraded by APP with Km and Vmax values comparable to those found for BK (Km = 19.7 +/- 2.6 microM; Vmax = 12.1 +/- 1.2 nmol/min/mL). In contrast, B2 antagonists containing D-Arg0 N-termini, including D-Arg[Hyp3,Thi5.8,D-Phe7]BK and D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, were resistant to APP-mediated hydrolysis. These data support a role for plasma aminopeptidase P in the degradation of circulating kinins, and a variety of B2 and B1 kinin agonists and antagonists. However, APP does not participate in the degradation of D-Arg0-containing antagonists.

Mechanism of digestion of bradykinin and lysylbradykinin (kallidin) in human serum. Role of carboxypeptidase, angiotensin-converting enzyme and determination of final degradation products.

Degradation of bradykinin and lysylbradykinin was studied in plasma and serum, and the results were compared to those seen with mixtures of carboxypeptidase N and angiotensin-converting enzyme (ACE), the two recognized kininases in blood. Angiotensin-converting enzyme was an effective kininase in mixtures with carboxypeptidase N at physiologic concentration and digested bradykinin to the dipeptides Phe- Arg and Ser-Pro plus the pentapeptide Arg-Pro-Pro-Gly-Phe. Carboxypeptidase N slowly removed the C-terminal Arg from bradykinin to yield des-Arg9-bradykinin (DBK); the latter was digested by ACE to yield the aforementioned pentapeptide and the tripeptide Ser-Pro-Phe. In serum, however, the C-terminal Arg was removed from bradykinin about five times faster than was accounted for by the activity of carboxypeptidase N. The primary substrate of ACE in serum, therefore, was des-Arg9-bradykinin and not bradykinin. The products of this reaction, pentapeptide and tripeptide, were unstable in serum and were cleaved by enzymes that have not yet been characterized. One product, free phenylalanine, was used to monitor these reactions by HPLC. Our studies indicate that the final products of bradykinin degradation were the tripeptide Arg-Pro-Pro, one mole each of Ser, Pro, Gly, and Arg, and two moles of phenylalanine. Since the serum level of carboxypeptidase N did not account for the rapid kinin degradation seen, other carboxypeptidases may have been operative, perhaps released as a result of blood clotting, or a serum cofactor may augment carboxypeptidase N activity.

Identification of [hydroxyproline3]-lysyl-bradykinin released from human plasma protein by kallikrein.

[Hydroxyproline3]-lysyl-bradykinin [( Hyp3]-Lys-BK), a new kinin was isolated, besides lysyl-bradykinin (Lys-BK), from the reaction mixture of human plasma protein Cohn's fraction IV-4 with hog pancreatic kallikrein. The liberated kinins were isolated by procedures including ethanol extraction, Sephadex G-15, CM cellulose and reverse-phase high performance liquid chromatography (HPLC) and quantitated by radioimmunoassay. On HPLC, two peaks of immunoreactive kinins emerged. Peak 1, an unknown kinin proceeded to Peak 2 which had an identical retention time to that of Lys-BK. The amino acid sequence of the unknown Peak 1 proved to be Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg, or [Hyp3]-Lys-BK, and Peak 2 Lys-BK. The ratio of the amounts of two kinins thus formed were [Hyp3]-Lys-BK 25 +/- 4% and Lys-BK 75 +/- 4%. The existence of [Hyp3]-Lys-BK suggests a presence of a new kininogen, containing [Hyp3]-Lys-BK in human plasma protein, possibly undergone post-translational modifications.