Influence of the centrifuge time of primary plasma tubes on routine coagulation testing.

Preparation of blood specimens is a major bottleneck in the laboratory throughput. Reliable strategies for reducing the time required for specimen processing without affecting quality should be acknowledged, especially for laboratories performing stat analyses. The present investigation was planned to establish a minimal suitable centrifuge time for primary samples collected for routine coagulation testing. Five sequential primary vacuum tubes containing 0.109 mol/l buffered trisodium citrate were collected from 10 volunteers and were immediately centrifuged on a conventional centrifuge at 1500 x g, at room temperature for 1, 2, 5, 10 and 15 min, respectively. Hematological and routine coagulation testing, including prothrombin time, activated partial thromboplastin time and fibrinogen, were performed. The centrifugation time was inversely associated with residual blood cell elements in plasma, especially platelets. Statistically significant variations from the reference 15-min centrifuge specimens were observed for fibrinogen in samples centrifuged for 5 min at most and for the activated partial thromboplastin time in samples centrifuged for 2 min at most. Meaningful biases related to the desirable bias were observed for fibrinogen in samples centrifuged for 2 min at most, and for the activated partial thromboplastin time in samples centrifuged for 1 min at most. According to our experimental conditions, a 5-10 min centrifuge time at 1500 x g may be suitable for primary tubes collected for routine coagulation testing.     

Induced hypoglycemia. A unusual case of child battering

We describe a case of Munchausen's syndrome by proxy in a 12 years old child. The administration of glibenclamide by the mother led to severe hypoglycemias in the child, who underwent various instrumental researches and a subtotal pancreatectomy before the final diagnosis could be reached. The diagnosis also was possible with the substantial help of an accurate psychological survey. The case solution, with disappearance of hypoglycemias, was made possible by the removal of the maternal presence settled by the juvenile court.     

Buprenorphine in balanced anesthesia

Buprenorphine was administered for the balanced general anaesthesia in 300 patients during several operations. The results obtained, the good features of analgesia, morphine-like, the absence of cardiovascular and respiratory side effects are presented.     

Spatiotemporal modeling of cerebral evoked magnetic fields to median nerve stimulation.

We measured somatosensory evoked magnetic fields during median nerve stimulation in 6 normal subjects. We applied multiple dipole models to study the spatiotemporal structure of early somatosensory evoked magnetic fields (SEFs), as well as the number, 3-dimensional location and time activity of their underlying neuronal sources. Two dipole sources were necessary to model the first 40 msec of SEFs explaining 85% of the data variance. Source 1 was located deeper than source 2, showed primarily a tangential orientation, and accounted for a larger part of the variance; source 2 showed no consistent orientation across subjects. Both sources showed biphasic time activities corresponding to the previously described N20-P30 and P25-N35 components. Spatiotemporal modeling could identify sources which could not be modeled consistently above noise by single moving dipoles (P25 component), revealed small latency differences of the two sources in some subjects suggesting parallel activation of these sources, and allowed separation of sources overlapping considerably both in space and time. We conclude that spatiotemporal modeling of SEFs may be useful to study functional anatomy of human sensorimotor cortex non-invasively.     

Coupling of lactose molecules to the carrier protein hinders the spleen and bone marrow uptake of doxorubicin conjugated with human albumin.

Several attempts have been made to enhance doxorubicin (DOXO) concentrations in tumour cells by drug conjugation with human albumin (HSA). HSA-DOXO has the drawback of causing DOXO accumulation in spleen and bone marrow, with a consequent leucopoenia not produced when lactose molecules are coupled to the carrier protein. In the present experiments we demonstrated that the effect of HSA lactosamination is not a consequence of a more rapid disappearance from the bloodstream of the lactosaminated conjugate (L-HSA-DOXO), which is rapidly internalized by the liver through the asialoglycoprotein receptor, but is due to a hindered uptake by spleen and bone marrow cells caused by the coupled lactose molecules. Experiments in vitro showed that HSA-DOXO produced an inhibition of murine macrophage proliferation not caused by L-HSA-DOXO. This result can be explained by higher amounts of the former conjugate entering in these cells and suggests macrophages as the cell type responsible for the spleen and bone marrow internalization of HSA-DOXO hindered by lactose coupling. Importantly, lactosamination of HSA did not reduce the marked uptake of HSA-DOXO by chemically induced rat hepatocellular carcinoma. L-HSA-DOXO, by avoiding DOXO accumulation in bone marrow is an attractive candidate for clinical trials against tumors which were found to actively internalize this conjugate in laboratory animals, such as hepatocellular carcinoma.     

BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

Comparison of glycaemic control over 1 year with pioglitazone or gliclazide in patients with Type 2 diabetes.

AIMS: To compare long-term (1 year) efficacy and safety of pioglitazone and gliclazide in patients with Type 2 diabetes. METHODS: This was a double-blind, multicentre, comparative, parallel group trial in 283 patients with Type 2 diabetes, who were randomized to receive 1-year treatment with pioglitazone 30-45 mg/day or gliclazide 80-320 mg/day. Drug dose was titrated on the basis of self-monitored blood glucose (SMBG) measurements and HbA1c values. The 1-year changes in HbA1c, fasting blood glucose (FBG), insulin, HOMA-S (HOmeostatic Model Assessment) and SMBG were compared. In a subgroup of patients (n = 10), systemic glucose production and utilization were determined by a combination of isotopic (deuterated glucose) and clamp techniques. RESULTS: In both groups, there were similar decreases in HbA1c (pioglitazone: -0.79%; gliclazide: -0.79%) and FBG (pioglitazone: -1.0 mmol/l; gliclazide: -0.7 mmol/l), whereas the slope of the reduction of fasting blood glucose was different between groups (P = 0.004). Insulin levels as well as insulin resistance assessed using HOMA-S decreased significantly only after pioglitazone treatment (-11.94 pmol/l and -1.03, respectively, both P = 0.002 vs. baseline). A significantly greater reduction in systemic glucose production was observed in the pioglitazone group (-2.48 micromol/kg/min, P = 0.042) than in the gliclazide group (-1.02 micromol/kg/min). A few, mild adverse events occurred in both groups. CONCLUSIONS: A comparable decrease in HbA1c and FBG was observed with pioglitazone and gliclazide. However, with pioglitazone there was a continuous decrease in FBG over 1 year, whereas gliclazide failed to maintain a similar trend. This favourable effect of pioglitazone was due to its insulin-sensitizing effect and ability to decrease systemic glucose production.     

Risks and complications of total parenteral nutrition. Personal experience

Total parenteral nutrition is now a broadly used method whose efficacy is confirmed, even if it is still not free from risk of complications. The Authors report their own clinical experience on 130 cases in the period 1981-1988. Complications were registered in 4.6% of the observed cases. They underline the problems relating to this subject.