Oxygen delivery enhancers: past, present, and future.

In endurance sport the delivery of oxygen to muscles plays a critical role. Indeed, muscle performance declines during prolonged and intense activity as a consequence of the shift from the aerobic to the anaerobic metabolism with an increase of lactate. To enhance the aerobic capacity 2 alternatives may be used: increasing either the transport or the delivery of oxygen. In this setting, blood doping is the practice of illicitly using a drug or blood product to improve athletic performance. Based on this definition, blood doping techniques may include: 1) blood transfusion (autologous or omologous); 2) erythropoiesis-stimulating substances [recombinant human erythropoietin (alpha, beta, omega), darbepoietin-alpha, continuous erythropoiesis receptor activator, hematide]; 3) blood substitutes (hemoglobin-based oxygen carriers, perfluorocarbon emulsions); 4) allosteric modulators of hemoglobin (RSR-13 and RSR-4); 5) gene doping (human erythropoietin gene transfection); 6) gene regulation (hypoxia-inducible transcription factors pathway). In the present overview we will briefly describe the above-mentioned techniques with the aim of underlining potential hematological alternatives to gene doping for increasing aerobic capacity in sport.

Sport-related hyperhomocysteinaemia: a putative marker of muscular demand to be noted for cardiovascular risk.

OBJECTIVE: Regular physical activity is associated with a reduction of cardiovascular morbidity and mortality; however, evidence of unfortunate cardiovascular events accompanying elite sport involvement continues to accumulate. To date, no information is available on possible peculiarities of the cardiovascular risk profile in athletes. DESIGN: The aim of this study was to evaluate plasma homocysteine levels in a group of athletes and to search for relationship with vitamin status and other metabolic variables in order to confirm the existence of a "sport-related hyperhomocysteinaemia" and to explain its clinical significance. The study population was composed of 82 athletes (59 male and 23 female) practising different sports and 70 healthy age-matched subjects (40 male and 30 female) as a control group. Besides the general clinical and analytical determinations, the assessed variables included homocysteine, folate, vitamin B12, total and high-density lipoprotein (HDL) cholesterol, lactate dehydrogenase (LDH), creatine kinase (CPK) and interleukin-6 (IL-6). RESULTS: The prevalence of hyperhomocysteinaemia (>15 micromol/l) in athletes and controls was 47% and 15%, respectively. No correlation was found between homocysteine and any of the other investigated variables, in particular plasma folate, blood pressure, LDH, CPK, total and HDL cholesterol and IL-6. CONCLUSION: The results of this study confirm the existence of a sport-related hyperhomocysteinaemia which appears linked neither to the same variables found in the general population, nor to specific training-related variables. We suggest that it would represent an adaptation to training but the possibility of a secondary vascular damage cannot be excluded.

A peculiar pattern of expression of the transferrin receptor (CD71) by reticulocytes in patients given recombinant human erythropoietin (rHuEPO): a novel marker for abuse in sport?

To overcome the limitation of the currently adopted direct method to detect recombinant Human Erythropoietin (rHuEpo) abuse in sport, indirect analysis of blood parameters are increasingly used as part of the anti-doping strategies. The aim of the present work is to identify whether immunophenotype modifications on erythroid cells may be indicative of previous rHuEPO administration. The study was conducted on dialyzed patients under treatment with rHuEPO (DPT). Dialyzed patients without rHuEPO therapy (DP) and volunteer donors (H) were used as controls. The analysis of erythroid cells immunophenotype, performed using a multiparametric flow cytometry technique, showed a peculiar pattern of CD71 expression following rHuEPO treatment. In particular CD71 showed an increased expression in mature and intermediate reticulocytes and a surprisingly decreased expression in immature reticulocytes. In conclusion, the analysis of reticulocyte maturation stages with TO/CD71 double staining may be considered as a valid alternative indirect method for the detection of rHuEPO abuse.

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death.

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.

Dose-dependent inhibition of cell proliferation induced by lipid peroxidation products in rat hepatoma cells after enrichment with arachidonic acid.

Polyunsaturated fatty acids (PUFA) are important constituents of membrane phospholipids, whose levels are decreased in some tumor cells. This deficiency may cause alterations in signal transduction and an interruption of normal cellular events. The enrichment of tumor cells with PUFA may stimulate or inhibit tumor growth, probably depending on the type of PUFA and the cellular concentration of aldehydes derived from restored lipid peroxidation. We examined the effect of several doses of prooxidant on the growth of hepatoma cells with different aldehyde dehydrogenase activities, enriched with arachidonic acid. Two doses of prooxidant were sufficient to reduce growth of hepatoma cells with low aldehyde dehydrogenase activity, whereas three doses were necessary for those with high enzyme activity. In both cases, lipid peroxidation products blocked the cells in the S phase.

A biparametric flow cytometry analysis for the study of reticulocyte patterns of maturation.

Automated haematological analysers still represent the gold standard for the study of reticulocyte maturation even if this technique is based on structural properties and staining affinity rather than on functional aspects. On the contrary, flow cytometry allows the simultaneous analysis of multiple cellular characteristics including functional features. Aim was to investigate whether simultaneous analysis of different reticulocyte parameters using flow cytometry may add functional information when considering their pattern of maturation. Thirty-nine healthy donors (H) and 31 haemodialysed patients on treatment with rHuEpo (HDT) were analysed. Reticulocyte counts and their stages of maturation were studied both with ADVIA 2120 and by flow cytometry. TO/CD71 scattergraph reticulocyte analysis designed a peculiar distribution which was similar among the same group of subjects (H or HDT), but different between H and HDT. distribution of the percentage of reticulocytes in low, medium and high boxes calculated by ADVIA 2120 did not show any difference between H and HDT groups, while the analysis using flow cytometry pointed out statistically significant differences between H and HDT groups in the three boxes where the TO+/CD71+ reticulocytes were localized. The present study suggests that TO/CD71 analysis was reproducible and could detect different pattern of maturation of a particular clinical setting.

The affinity labeling of amino acids in or about the active center of DNA-dependent DNA polymerase I.

The use of an affinity label and an inhibitor that shows relative specificity for one amino acid has led to the identification of two amino acid residues in or near the active center of DNA-dependent DNA polymerase I. [35S]-beta-D-Ribosyl-6-methylthiopurine periodate oxidation product ([35S]MMPR-OP) and [14C]phenylglyoxal ([14C]PG) were used to elucidate the presence of a single lysine and arginine in or about the active center of the enzyme.

Increase in class 2 aldehyde dehydrogenase expression by arachidonic acid in rat hepatoma cells.

Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPAR alpha expression, whereas PPAR gamma expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.