Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia.

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for alpha-globin over that for beta-globin and intact HbA in an adenosine 5'-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the alpha-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in alpha-globin compared with intact haemoglobin, HbA and the beta-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the alpha-globin chain over that of the beta-globin in the haemoglobin disorder beta-thalassaemia.

Is subdiaphragmatic aortic cross-clamping a suitable model for spinal cord ischemia/reperfusion injury study in rats?

OBJETIVO: Avaliar a eficácia do pinçamento da aorta subdiafragmática no modelo experimental de isquemia/reperfusão da medula espinhal em ratos. Trinta e seis ratos Wistar, machos, foram aleatoriamente distribuídos em 2 grupos (n=18) e submetidos ao pinçamento subdiafragmático da aorta, durante 30 minutos (Grupo-2 -Isquemia/Reperfusão). Os ratos do Grupo-1 (G-1 - Sham) foram utilizados como controles e submetidos a laparotomia sem pinçamento arterial. As amostras (medula e sangue arterial) foram coletadas ao término do período de isquemia (T-0) e 10 (T-10) e 20 (T-20) minutos mais tarde e nos mesmos intervalos, no grupo G-1. As concentrações teciduais e sanguíneas de piruvato, lactato, glicose e as concentrações medulares de trifosfato de adenosina (ATP) foram determinadas por ensaios enzimáticos. RESULTADOS: As concentrações de piruvato e glicose (sangue e tecido) e de lactato e ATP (medula) não foram diferentes,comparando G-1 versus G-2. A lactacemia elevou-se significantemente no G-2, comparado ao G-1, durante a reperfusão (T-10). CONCLUSÃO: O modelo experimental de pinçamento subdiafragmático da aorta não é adequado para o estudo da lesão de isquemia/reperfusão na medula de ratos, uma vez que não proporciona alterações nas concentrações in vivo de metabólitos teciduais, por exemplo de lactato ou ATP, compatíveis com aquelas encontradas em tecidos sujeitos à isquemia/reperfusão.

Metabolismo miocárdico após cardioplegia sangüínea hipotérmica retrógrada contínua com inducão anterógrada normotérmica / Myocardial metabolism after hypothermic retrograde continuous blood cardioplegia with anterograde warm cardioplegic induction

OBJETIVO: Determinar as alteracões sofridas pelo miocárdio durante a cardioplegia sangüínea hipotérmica retrógrada contínua com a adicão da inducão cardioplégica anterógrada normotérmica. MÉTODO: Análise metabólica da cardioplegia sangüínea hipotérmica retrógrada contínua com inducão anterógrada normotérmica em estudo prospectivo de 15 pacientes consecutivos. Amostras de sangue arterial e do seio coronário foram simultaneamente colhidas para análise do conteúdo de oxigênio e da concentracão de lactato. Quatro biópsias miocárdicas foram obtidas para análise dos níveis de ATP, ADP, AMP e lactato no miocárdio. A isoenzima CK-MB foi analisada no sangue venoso. RESULTADOS: Não houve mortalidade no grupo. Nenhum paciente necessitou de suporte inotrópico na saída de CEC e não foi detectado IAM per ou pós-operatório. Ocorreu diminuicão da extracão artério-venosa do lactato e do oxigênio pelo coracão durante a reperfusão, havendo uma recuperacão parcial ao final de 60 minutos de reperfusão. Os níveis miocárdicos de ATP e de seus nucleotídeos foram mantidos durante o pincamento aórtico, porém houve reducão destes nos primeiros 30 minutos de reperfusão. O lactato acumulou-se no músculo cardíaco durante o pincamento aórtico, havendo reducão durante a reperfusão. CONCLUSÕES: Concluímos por uma análise metabólica que o método não conseguiu evitar o metabolismo anaeróbico durante o período de pincamento aórtico e que somente com 60 minutos de reperfusão foi observado um grau de recuperacão metabólica satisfatória. Provavelmente essas alteracões são devido à injúria isquêmica celular ocorrida durante o pincamento aórtico e parecem ter efeito transitório. Observamos melhora da protecão miocárdica com o acréscimo da inducão cardioplégica anterógrada normotérmica.

Evaluation of platelet storage lesions in platelet concentrates stored for seven days.

BACKGROUND & OBJECTIVES: Platelet storage lesions (PSL) are detrimental to the post transfusion functional capacity of platelets. As EDTA enhances the storage-induced changes, changes in platelet indices with and without EDTA incubation are promising new tests to monitor the PSL. The present study was undertaken to monitor the PSL in 40 units of pooled platelet concentrates harvested by platelet rich plasma (PRP) method and stored for seven days in second generation platelet storage containers using conventional in vitro tests and platelet indices. METHODS: Morphological changes in platelets were monitored by automated haematological cell counter for platelet count and mean platelet volume (MPV). Samples were incubated with K2EDTA for 1 h and platelet indices were repeated on the EDTA incubated samples. Difference between pre-and post-EDTA incubation of platelet count (dPLT) and MPV (dMPV) were calculated. Metabolic parameters such as pH, pO2, pCO2 and ATP were measured. RESULTS: There was no significant change in the indices without EDTA during storage, however, after EDTA incubation, significant changes were noted in dPLT and dMPV. The mean dPLT on day 0 was 75.15 x 10(3)/microl decreasing to 44.4 x 10(3)/microl on day 7, while dMPV from 0.76 fl on day 0 increased to 1.34 fl on day 7 (P < 0.05). Metabolic parameters showed a significant decrease in pH and pCO2 concurrent with increasing pO2 during storage (P < 0.05). Average ATP level on day 0 was 21.09 micromol/dl falling to 10.59 micromol/dl on day 7. INTERPRETATION & CONCLUSION: The results indicate that storage induced lesions take place even in second generation platelet storage containers under recommended conditions of storage. Platelet indices especially after EDTA incubation are useful in monitoring PSL. However, how much these changes contribute to poor post transfusion survival and haemostatic function of platelets need to be investigated.

Serum ATP level: a useful measure for fatigue in patients with rheumatoid arthritis

Fatigue is a frequently occurring symptom in individuals with rheumatic diseases, including rheumatoid arthritis [RA], and contributes to many disabilities in those patients. Several variables acting singly or in combination may contribute to the mechanism of this fatigue. The end result of these factors is the depletion of intracellular and extracellular adenosine triphosphate [ATP]. This study was conducted to assess ATP levels in patients with active and inactive RA, and its correlation to fatigue. Forty-two RA patients were chosen from the outpatient clinics of Al-Azhar University Hospitals, over two years duration [1999-2000]. Patients with suspected or evidenced to have myositis, or under corticosteroid therapy were excluded from the study. The patients were classified into two groups: active RA [22 patients], and inactive RA [20 patients]. Another group of 20 normal volunteer subjects was selected as a control group. RA disease activity, the short-form McGill Pain Questionnaire including visual pain analogue scale [VAS], Ritchie's articular index, early morning stiffness, fatigue visual analogue scale [FVAS], and routine laboratory tests were all measured. Serum ATP was measured by modified enzymatic UV test. Early morning stiffness, Ritchie's articular index, hemoglobin level, pain score, and erythrocyte sedimentation rate, were all increased significantly in active and inactive RA than controls, and were not correlated to fatigue. Serum ATP level was significantly lowered in active than inactive RA and controls, while it was not significantly different between inactive RA and normal controls, and it was highly significantly inversely correlated to fatigue as measured by the fatigue- visual analogue scale. The overall outcome of fatigue processes is the depletion of ATP stores, with resultant lowered intracellular and extracellular levels of ATP. Serum ATP level was the only reliable and precise factor for measuring fatigue in RA patients

Is it antemortem or postmortem blood?

Bloodstains examination is required in many fields in medicolegal practice. This study was carried out to discriminate between antemortem [AM] andpostmortem [PM] bloodstains. The method used depended on the determination ofthe level of human myoglobin [by radioimmunoassay] as well as the levels ofadenosine triphosphate [ATP], xanthine and uracil [by HPLC] in thebloodstains. The study revealed a highly statistically significant elevationof myoglobin levels in PM than AM bloodstains. ATP was found in highconcentrations in all AM bloodstain samples and could not be detected in anyof the PM bloodstain ones. On the other hand, xanthine and uracil werepresent in considerable concentrations in all the PM bloodstain samples andwere not detected in any of the AM bloodstain ones. These results wereindependent of the age of the stain, the postmortem interval and the cause ofdeath. It was suggested that the determination of the above-mentionedparameters could help in the discrimination between antemortem and postmortembloodstains in medicolegal practice

Synthesis of NAD+ in erythrocytes incubated with nicotinic acid and the effect of di-(2-ethyl hexyl) phthalate (DEHP).

Synthesis of NAD+ from nicotinic acid by erythrocytes incubated in SAGM phosphate solution and effect of di-[2-ethyl hexyl] phthalate, a plasticizer commonly used in PVC blood/component storage bags, on this synthesis was studied. Erythrocytes are able to synthesise NAD+ in SAGM (sodium chloride, adenine, glucose, mannitol) phosphate solution and this synthesis was more in the presence of added nicotinic acid (optimum concentration 1 mM). The level of NAD+ decreased when the incubation period was increased from 24 to 48 hr. Glutamine had a deleterious effect on this synthesis, possibly due to the decrease in pH. Di-[2-ethyl hexyl] phthalate had an inhibitory effect on NAD+ synthesis when the cells were incubated in SAGM phosphate solution, either alone or in the presence of added nicotinic acid. There was significant decrease in the release of potassium and haemoglobin from the cells in the presence of nicotinic acid, indicating increased red cell stability.

Correlation of discocyte frequency and ATP concentration in preserved blood: a morphological indicator of red cell viability

Red blood cells (RBC) are viable if kept in an adequate preservative solution, although gradual changes in morphology and metabolism may occur. There is a gradual decrease in adenosine-5'-triphosphate (ATP) cocentration, pH, glucose consumption, and enzyme activity during preservation. The normal discocyte shapes are initially replaced by echimocytes and stomatocytes and, at final stages, by spherocytes, the last before eplenic sequestration. Post-transfusional survival has been correlated with the ATP concentration. RBC preserved in ADSOL, a solution containing adenine, dextrose, sodium chloride, and mannitol, are viable for transfusion for up to 6 weeks. Erythrocytes from 10 blood units taken from healthy adult donors were preserved for 12 weeks in ADSOL at 4 graus Celsius. We now report a significant correlation (r2 = 0.98) between the percentage of discocytes (89 to 7 percent) and ATP (100 to 10 percent) concentration in ADSOL-preserved RBC. The results suggest that the percent of discocyte shapes used as an indicator OF ATP concentration may be a useful indicator for quality control of RBC viability in centers which have limited assay facilites.