BACKGROUND: The objective of this study was to evaluate the role of proteases on the degradation of parathyroid hormone (PTH) in blood samples. METHODS: Protease inhibitors with specificity against serine proteases (aprotinin), cysteine proteases (E-64), serine and cysteine proteases (leupeptin), metalloproteases (EDTA), or a protease inhibitor cocktail with a broad spectrum of inhibitory activity were added to blood samples. After storage at room temperature (0-48 hr), PTH levels were measured. RESULTS: PTH levels in samples with the protease inhibitor cocktail did not change significantly after 48 hr of storage at room temperature, but the average PTH levels decreased by 40.7% and 20.1%, in samples stored at room temperature and stored at 4degrees C without protease inhibitors, respectively. PTH levels in samples with leupeptin were stable for up to 24 hr. After 48 hr, the mean PTH levels decreased by 17.1%, 16.0%, 26.2%, and 32.1%, with 500 KIU/mL aprotinin, 100 micro mol/L leupeptin, 10 micro mol/L E-64, and 10 micro mol/L EDTA, respectively, in the samples stored at room temperature. CONCLUSIONS: The decrease in PTH levels in blood samples seemed to be due to the degradation of PTH by proteases. Various proteases, including especially serine proteases, would act together to degrade PTH in blood specimen. The PTH degradation may be inhibited in blood specimen with protease inhibitor cocktail.
Aprotinin/pharmacology ; Blood Specimen Collection ; Edetic Acid/pharmacology ; Female ; Humans ; Leucine/analogs & derivatives/pharmacology ; Leupeptins/pharmacology ; Male ; Parathyroid Hormone/*blood/metabolism ; Protease Inhibitors/*pharmacology ; Time Factors

BACKGROUNDInflammation and coagulation are two intimately cross-linked defense mechanisms of most, if not all organisms to injuries. During cardiopulmonary bypass (CPB), these two processes are activated and interact with each other through several common pathways, which may result in subsequent organ dysfunction. In the present study, we hypothesized that the addition of nitric oxide, prostaglandin E1 (PGE1), and aprotinin to the systemic circulation, hereby referred to as blood hibernation, would attenuate the inflammation and coagulation induced by CPB.

METHODSThirty adult mongrel dogs were equally divided into five groups, anesthetized and placed on hypothermic CPB (32 degrees C). Each group received respectively the following treatments: (1) inhalation of 40 ppm nitric oxide; (2) intravenous infusion of 20 ng x kg(-1) x min(-1) of PGE1; (3) 80,000 kallikrein inhibitor units (KIU)/kg of aprotinin; (4) the combination of all three agents (blood hibernation group); and (5) no treatment (control group) during CPB. Activation of leukocyte, platelet, endothelial cell, and formation of thrombin were assessed after CPB.

RESULTSAs compared with the other four groups, leukocyte counts were higher, while plasma elastase, interleukin-8, CD11b mRNA expression, myeloperoxidase activities and lung tissue leukocyte counts were lower in the blood hibernation group (P < 0.05 versus other four groups after CPB). Plasma prothrombin fragment (PTF)1+2, and platelet activation factors were lower, while platelet counts were higher in the blood hibernation group (P < 0.05 versus other four groups at 6 and 12 hours after CPB). Electron microscopy showed endothelial pseudopods protrusion, with cell adherence in all four groups except the blood hibernation group where endothelial cells remained intact.

CONCLUSIONBlood hibernation, effected by the addition of nitric oxide, PGE1 and aprotinin to the circulating blood during extra-corporeal circulation, was observed to attenuate the inflammation and coagulation induced by cardiopulmonary bypass, most likely by inhibiting the important common intermediates between the two cross-linked processes.

Alprostadil ; pharmacology ; therapeutic use ; Animals ; Aprotinin ; pharmacology ; therapeutic use ; Blood Coagulation ; drug effects ; CD11b Antigen ; genetics ; Cardiopulmonary Bypass ; adverse effects ; Dogs ; Inflammation ; drug therapy ; etiology ; Male ; Nitric Oxide ; pharmacology ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction

Pulmonary vascular resistance (PVR) is generally believed to be elevated after cardiopulmonary bypass (CPB) due to whole body inflammation. Aprotinin has an antiinflammatory action, and it was hypothesized that aprotinin would attenuate the PVR increase induced by CPB. Ten mongrel dogs were placed under moderately hypothermic CPB for 2 hr. The experimental animals were divided into a control group (n=5, group I) and an aprotinin group (n=5, group II). In group II, aprotinin was administered during pre-bypass (50,000 KIU/kg) and post-bypass (10,000 KIU/kg) periods. Additional aprotinin (50,000 KIU/kg) was mixed in CPB priming solution. PVRs at pre-bypass and post-bypass 0, 1, 2, 3 hr were calculated, and lung tissue was obtained after the experiment. Post-bypass PVRs were significantly higher than prebypass levels in all animals (n=10, p<0.001). PVR elevation in group II was less than in group I at 3 hr post-bypass (p=0.0047). Water content of the lung was lower in group II (74+/-9.4%) compared to that of group I (83+/-9.5%), but the difference did not reach significance (p=0.076). Pathological examination showed a near normal lung structure in group II, whereas various inflammatory reactions were observed in group I. We concluded that aprotinin may attenuate CPB-induced PVR elevation through its anti-inflammatory effect.
Animals ; Aprotinin/*pharmacology ; *Cardiopulmonary Bypass ; Comparative Study ; Dogs ; Hemostatics/pharmacology ; Lung/*blood supply/metabolism/pathology ; Male ; Models, Animal ; Research Support, Non-U.S. Gov't ; Vascular Resistance/*drug effects ; Water/metabolism

BACKGROUND: Post-CPB bleeding diathesis remains one of main causes of morbidity in open heart operation since it requires significant amount of homologous transfusion. Many approaches have been studied to reduce the amount of postoperative blood loss and blood use. Aprotinin is known to reduce postoperative bleeding. Therefore, hemostatic effects of aprotinin have been searched in open heart surgery. METHODS: Adult patients for open heart opeartion were randomly allocated to two groups, control group and study group. All anesthetic, surgical and bypass techniques were standardized. After induction, 10,000 KIU given for testing and then 2,000,000 KIU of aprotinin was infused for 20~30 minutes before sternotomy, and followed by at rate of 500,000 KIU/hr. Additional 2,000,000 KIU of aprotinin was mixed to pump prime of heart-lung machine. Aprotinin infusion was ended by the time of skin closure. Amount of postoperative bleeding as well as homologous blood requirement were measured. Urine output and serum creatinine were also documented during postoperative period. RESULTS: Postoperative-36hr summation of blood loss differed significantly(1858.4 1182.9 ml vs. 1256.7 688.4 ml, p<0.1). Postoperative homologous blood requirements were also reduced in the requirement of packed RBCs use(3.0 2.5 units vs. 1.0 0.8 units, p<0.01) and of fresh frozen plasma use(6.6 4.4 units vs. 3.9 2.6 units, p<0.05). Postoperative measurement of urine output and serum creatinine showed insignificant difference between groups while remained within normal range. CONCLUSIONS: High-dose aprotinin seems to have beneficial effect in reducing postoperative blood loss and blood use without renal complication.
Adult* ; Aprotinin* ; Blood Platelets ; Cardiopulmonary Bypass ; Control Groups ; Creatinine ; Disease Susceptibility ; Heart ; Heart-Lung Machine ; Hemorrhage ; Humans ; Pharmacology ; Plasma ; Postoperative Hemorrhage ; Postoperative Period ; Reference Values ; Skin ; Sternotomy ; Thoracic Surgery*