Characterization of uronic-acid-rich inhibitor of calcium oxalate crystallization isolated from rat urine

Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Uronic-acid-rich protein (UAP), a glycoprotein with a molecular weight of approximately 35 kDa, is one such inhibitor. Here we report the characterization of UAP extracted from rat urine using three chromatographic steps including diethylaminoethanol (DEAE)-Sephacel, Sephacryl S-300 and Mono Q column and compare it with human UAP. The molecular weight of rat UAP (UAP_r) is similar to that of human UAP (UAP_h), being approximately 35 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid compositions are identical, they contain a high percentage of aspartic and glutamic acids and they react positively in the carbazole reaction, suggesting that they contain uronic acid. The inhibitory activities of UAP_h and UAP_r were assayed on a calcium oxalate crystallization system in vitro using [⁴⁵Ca]calcium chloride. Both exert a strong inhibition, suggesting that UAP_r, like UAP_h, plays an important role in preventing and reducing calcium oxalate crystallization in the urine. On Western blot analysis, both UAP_h and UAP_r immunoreact with inter--trypsin inhibitor (ITI) antibody. Nevertheless, using the Ouchterlony immunodiffusion technique, there was no precipitation line between ITI antibody and UAP. Therefore, we hypothesize that UAP is related to ITI and that they may have the same epitope but are not completely identical. We conclude that UAP belongs to the ITI superfamily of macromolecules which contribute to the regulation of the calcium oxalate crystallization process.     

Role of inter-α-inhibitor and its related proteins in urolithiasis. Purification of an inter-α-inhibitor related protein from the bovine kidney

Urine contains several macromolecules that inhibit calcium oxalate (CaOx) crystallization. Among them is bikunin, the light chain of most of the inter-α-inhibitor (IαI) family of glycoproteins. This study aimed to verify whether bikunin and other members of the IαI family are synthesized in the kidneys or derived exclusively from the plasma. Proteins extracted from homogenized bovine kidney were applied successively to three chromatographic steps on DEAE-Sephacel, Sephacryl S-300, and Mono Q column. The inhibitory activity was assayed using a CaOx crystallization system. The presence of IαI-related proteins was determined by␣electrophoresis and Western blotting. The results showed that kidney extract contained a 125-kDa protein that cross-reacted with anti-IαI antibodies. This protein inhibited CaOx crystallization efficiently. According to its molecular weight and immunoreaction with anti-IαI antibody, the 125-kDa protein could be pre-α-inhibitor. The latter is known to encompass a heavy chain and bikunin, which may explain its inhibitory activity against CaOx crystallization. Consequently, we hypothesize that kidneys may produce some IαI-related proteins that are involved in the inhibition of stone formation. Received: 18 February 1998 / Accepted: 9 July 1998     

Effects of skin infiltration with ropivacaine 0,75% on postoperative pain after caesarean section

OBJECTIVE: Evaluate the effects of skin infiltration with ropivacaine 0,75% on postoperative pain after caesarean section in the first 24h. PATIENTS AND METHODS: A prospective randomized double blind study was realized during three months in Auxerre Hospital. All ASA 1-2 patients presenting for elective caesarean section under spinal anesthesia were enrolled in the study. Drug addicts and patients with chronic pain were excluded from the study. The patients were randomly divided into two groups to receive skin infiltration 20 ml of ropivacaine 0,75% (Gr R) or skin infiltration of 20 ml of 0,9% saline solution. All patients received systematically propacetamol 1g per six hours and ketoprofen 50mg per six hours. Intravenous morphine titration was delivered to patients with a simple numerical scale greater or equal to three (SNS> or =3). Postoperative pain (SNS), morphine consumption and adverse reactions were compared. RESULTS: From July to September 2005, 42 patients were enrolled in the study. The SNS was lower in the Gr R. Total morphine consumption was reduced in the Gr R. The incidence of the adverse effects were higher in the Gr P. One case of parietal haematoma was detected in the Gr P, the evolution of which was favorable. DISCUSSION AND CONCLUSION: Skin infiltration of ropivacaine 0,75% is a simple technique able to reduce postoperative pain score and morphine consumption after caesarean section.     

Timing of tracheostomy and outcome of patients requiring mechanical ventilation

Objectives

To assess the impact of tracheostomy timing on outcome of critically ill patients requiring mechanical ventilation (MV).

Study design

Retrospective clinical study in a twelve beds intensive care unit (ICU).

Patients and methods

From January 2001 to June 2005, patients under MV who received tracheostomy were divided into 2 groups: early tracheostomy group when tracheostomy was performed before or on day 7 and late tracheostomy group when it was performed thereafter. We compared prevalence of nosocomial pneumonia, length of sedation, lengths of MV, length of stay in ICU, weaning from MV and mortality rates between the 2 groups.

Results

During this period of 4 years and half, 112 patients underwent tracheostomy, 62 of whom had early tracheostomy and 50 had late tracheostomy. Early tracheostomy was associated with significant reduction of length of sedation (10 ± 3 vs 17 ± 5 days, P < 0.001), length of MV (21 ± 19 vs 29 ± 17 days, P = 0.02) and length of stay in ICU (33 ± 22 vs 42 ± 18 days, P = 0.042). There were no differences in prevalence of pneumonia (21% for early tracheostomy group vs 31% for late tracheostomy group, P = 0, 13), weaning from MV (50 vs 36%, P = 0.19), and mortality rates between the 2 groups (38 vs 54%, P = 0.15).

Conclusion

This study demonstrated that early tracheostomy (≤ 7 days), was associated with shorter length of sedation, shorter duration of MV and shorter ICU length of stay, without affecting weaning from MV, prevalence of nosocomial pneumonia or survival.     

Iatrogenic oesophageal perforation following an imprevisible difficult tracheal intubation

The authors report a case of oesophageal perforation after difficult tracheal intubation. Interest of an early diagnosis and management of such complication are discussed.     

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998