Potential role of levocarnitine supplementation for the treatment of chemotherapy-induced fatigue in non-anaemic cancer patients.

Ifosfamide and cisplatin cause urinary loss of carnitine, which is a fundamental molecule for energy production in mammalian cells. We investigated whether restoration of the carnitine pool might improve chemotherapy-induced fatigue in non-anaemic cancer patients. Consecutive patients with low plasma carnitine levels who experienced fatigue during chemotherapy were considered eligible for study entry. Patients were excluded if they had anaemia or other conditions thought to be causing asthenia. Fatigue was assessed by the Functional Assessment of Cancer Therapy-Fatigue quality of life questionnaire. Treatment consisted of oral levocarnitine 4 g daily, for 7 days. Fifty patients were enrolled; chemotherapy was cisplatin-based in 44 patients and ifosfamide-based in six patients. In the whole group, baseline mean Functional Assessment of Cancer Therapy-Fatigue score was 19.7 (+/-6.4; standard deviation) and the mean plasma carnitine value was 20.9 microM (+/-6.8; standard deviation). After 1 week, fatigue ameliorated in 45 patients and the mean Functional Assessment of Cancer Therapy-Fatigue score was 34.9 (+/-5.4; standard deviation) (P<.001). All patients achieved normal plasma carnitine levels. Patients maintained the improved Functional Assessment of Cancer Therapy-Fatigue score until the next cycle of chemotherapy. In selected patients, levocarnitine supplementation may be effective in alleviating chemotherapy-induced fatigue. This compound deserves further investigations in a randomised, placebo-controlled study.

H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis.

H2O2 treatment on U937 cells leads to the block of glycolytic flux and the inactivation of glyceraldehyde-3-phosphate-dehydrogenase by a posttranslational modification (possibly ADP-ribosylation). Glycolysis spontaneously reactivates after 2 h of recovery from oxidative stress; thereafter cells begin to undergo apoptosis. The specific ADP-ribosylation inhibitor 3-aminobenzamide inhibits the stress-induced inactivation of glyceraldehyde-3-phosphate-dehydrogenase and the block of glycolysis; concomitantly, it anticipates and increases apoptosis. Exogenous block of glycolysis (i.e., by culture in glucose-free medium or with glucose analogs or after NAD depletion), turns the transient block into a stable one: this results in protection from apoptosis, even when downstream cell metabolism is kept active by the addition of pyruvate. All this evidence indicates that the stress-induced block of glycolysis is not the result of a passive oxidative damage, but rather an active cell reaction programmed via ADP-ribosylation for cell self-defense.

Overexpression of erythrocyte glutathione S-transferase in uremia and dialysis.

BACKGROUND: Overexpression of glutathione S-transferase (GST; EC 2.5. 1.18) has been documented in the erythrocytes of patients with chronic renal failure, and this event may well be of relevance from a clinical standpoint. In fact, it could serve as a marker of uremic toxicity overall, which can contribute to impair the function and survival of the erythrocytes. However, the biochemical details of this phenomenon are poorly understood. METHODS: In this study, we characterized the expression of GST in erythrocytes of 118 uremic patients under different clinical conditions. The mechanisms responsible for the regulation of protein expression and enzyme activity were investigated in light of different dialysis approaches, oxidative stress, uremic toxins, erythrocyte age, and erythropoietin (EPO) supplementation. RESULTS: Mean GST activity in uremic patients was highly overexpressed with respect to controls, and this phenomenon was exclusively attributable to an increased expression of GST. Overexpression of GST did not appear to be dependent on oxidative stress and was not influenced by vitamin E supplementation. In the same manner, both erythrocyte age and EPO supplementation apparently did not interfere with the GST concentrations, which were the same in controls and patients. Preliminary experiments suggested that high-molecular weight or protein-bound toxins could play some role in the overexpression of GST. CONCLUSIONS: GST expression may be a useful marker for the individual accumulation of uremic toxins as well as of the efficiency of new dialysis strategies in removing them.

S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase decreases the enzyme affinity to the erythrocyte membrane.

The effects of nitric oxide (NO) or related molecules on the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the red blood cell (RBC) membrane were investigated. It was demonstrated that submillimolar concentrations of the NO donor sodium nitroprusside (SNP) not only strongly inactivated GAPDH by S-nitrosylation of the enzyme thiols but also decreased the binding affinity of GAPDH for the RBC membrane. In fact, the incubation with SNP for 60 min at 30 degrees C and at a concentration > 50 microM induced the dissociation of the native GAPDH from the white unsealed membranes (standard ghosts) in a concentration-dependent manner with a partial recovery of the enzyme activity and thiols when SNP concentrations higher of 1 mM were used. Binding experiments under saturating conditions indicate a Ka value for the nitrosylated GAPDH of 3.5 +/- 0.8 x 10(6) M-1, which was more than 50% less than the Ka value of 7.6 +/- 0.6 x 10(6) M-1 observed for the native enzyme. These data were also confirmed in reassociation experiments under nonsaturating conditions. Dithiothreitol (DTT), which at concentrations of less than 1 mM catalyzed the S-nitrosylation of GAPDH and the consequent modification of the binding properties described above, the concentrations higher than 5 mM restored both the enzyme activity and the binding properties. Furthermore, the enzyme-membrane association induced before the incubation step afforded at least partial protection from the loss of titrable thiols and from the inactivation induced either spontaneously or by SNP. Taken together, these data not only confirm the key role of the active site cysteine residues in the catalytic function of GAPDH but also suggest that they may be involved in the NO-dependent regulation of GAPDH binding to the RBC membrane.

Bioreactivity and biocompatibility of a vitamin E-modified multi-layer hemodialysis filter.

BACKGROUND: The present study was designed to test the biocompatibility of a new vitamin E-modified multi-layer membrane (CL-E filter), as well as its ability to protect against oxygen free radicals during hemodialysis (HD). METHODS: We investigated, both in vitro and in vivo, the bioreactivity of the filter with respect to the blood antioxidants and its ability to prevent lipoperoxidation. The effects on the leukocyte respiratory burst were also studied. Cuprammonium rayon was used as a comparison material (CL-S filter). RESULTS: The in vitro results demonstrated that, under controlled conditions, CL-E is able to preserve blood antioxidants, and particularly vitamin E, from the spontaneous consumption observed in the incubation with CL-S filters and in control incubations. In accordance with this observation, the rate of the oxidative demolition of lipids either in plasma and red blood cells (RBC) or from rat brain homogenate decreased after the exposure to CL-E filters in comparison with the CL-S filter. Moreover, in the absence of any significant cytotoxic effects due to both the types of material studied, the production of oxygen free radicals and nitric oxide (NO) by leukocytes was higher after their in vitro exposure to CL-S, but was quite similar to that of the control leukocytes after exposure to CL-E. In vivo, a one-month treatment with the CL-E filter increased plasma vitamin E by 84.3% with respect to treatment with CL-S; this gain slightly decreased to 68.9% when CL-E treatment was prolonged to three months. In the RBC, vitamin E was found to have increased by 76.7% and 113.4% at one and three months, respectively. Plasma glutathione (GSH) levels determined at three months were significantly increased from 0.10 +/- 0.02 to 0.33 +/- 0.12 mumol/ml, while the erythrocyte GSH was only slightly increased. The leukocyte function estimated as responsiveness to soluble chemical stimuli in CL-S-treated patients was significantly improved both qualitatively and quantitatively after CL-E treatment. The presence of an increased number of mononuclear cells undergoing programmed cell death (apoptosis) in CL-S-treated patients (18.8 +/- 1.7% vs. a control value of 6.5 +/- 2.3%) as well as the apoptogenic effect of their plasma in vitro on U937 cells was significantly corrected after CL-E treatment (mean decrease in apoptotic mononuclear cells at 24 hours of culture, 25.5% and 27.1% at 1 and 3 months, respectively). The anti-apoptogenic effect of CL-E treatment showed a close dependence on the increase in vitamin E in the blood cell compartment. CONCLUSIONS: This study suggests that this vitamin E-modified membrane can be considered a highly biocompatible material, the antioxidant properties of which can exert a site-specific and timely scavenging function against oxygen free radicals in synergy with a hypostimulatory action on the PMN respiratory burst.

Oxidative damage during hemodialysis using a vitamin-E-modified dialysis membrane: a preliminary characterization.

A comparison of the oxyradical exposure during hemodialysis (HD) carried out with vitamin-E-modified cellulose (CL-E) or conventional membranes, studying red blood cell (RBC) and plasma lipoperoxidation and RBC glutathione metabolism, was done. In this preliminary characterization of a new and original approach to the prevention of free radical damage in HD, the results obtained indicate that lipoperoxidation in plasma and RBC is decreased and therefore oxidative damage can be significantly decreased using CL-E dialysis membranes instead of conventional membranes.

Bicarbonate versus lactate buffer in peritoneal dialysis solutions: the beneficial effect on RBC metabolism.

OBJECTIVE: Using the erythrocyte as a model for other kinds of cells not directly exposed to peritoneal dialysis (PD) solutions, we investigated the tolerance of the cell metabolism to lactate and bicarbonate buffers. DESIGN: We studied, in vivo (in two groups of 5 PD patients each) and in vitro, the Embden-Meyerhof pathway (EMP) because it represents a potential target for the unphysiological effects of lactate or bicarbonate buffers. The EMP is the main glucose-utilizing route in the red blood cell (RBC), producing energy and reducing power. METHODS: The enzymatic activities of the key steps in the glycolytic pathway and the energy charge (EC), determined by the levels of phosphorylated adenine nucleotides, were investigated spectrophotometrically and by high performance liquid chromatography (HPLC) in two groups of patients undergoing lactate (L-group) and bicarbonate (B-group) PD, respectively. The in vitro effects of both bicarbonate and lactate buffers on some EMP enzyme activities and energy production were determined. Cellular pH (pHi) was also investigated. RESULTS: The B-group showed an EC value near the control levels, while in the L-group a significantly lower EC value was observed (t-test: p < 0.05 vs both B-group and controls). The key enzymes in the EMP, and in particular hexokinase, were higher in the L-versus B-group (p < 0.03 for the comparison of the Hk mean values). As demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the bound form of glyceraldehyde-3-phosphate dehydrogenase (G-3-PD), an inactive form of this EMP enzyme, was significantly higher in the L-group with respect to the B-group (p < 0.004). In the in vitro experiments, high lactate concentrations acutely inhibited the key enzymatic steps of glycolysis, producing a significant decrease in glucose consumption and adenosine triphosphate production. These effects were not observed when bicarbonate was used in the incubations. Both in vivo and in vitro lactate, but not bicarbonate, induce a significant drop in pHi (p < 0.05). Decreased levels of pHi like those observed in the lactate-incubated RBC were demonstrated to be able to inhibit G-3-PD activity (25 +/- 2%) here used as an indicator of the actual decrease in pH. CONCLUSION: This study provides evidence for a damaging action of lactate with respect to bicarbonate buffer on the RBC metabolism. This condition was demonstrated observing a cell energy depletion, which coincides in vitro with an acute EMP impairment; the lactate accumulation together with the consequent lowering of pHi seem to be responsible for this effect, which was not observed when bicarbonate was used instead of lactate.

Morphological alterations and increased resistance to hemolysis in t-butyl hydroperoxide incubated RBC from elderly subjects.

The response of human red blood cells (RBC) to oxidative stress has been studied with the aim to evaluate any difference in the behavior of cells from young and old subjects. Thus, RBC from 5 young (27 +/- 2 years) and 5 old (80 +/- 5 years) individuals have been treated with the organic peroxide t-butyl hydroperoxide (TBHP). The two groups behaved differently: after 4 hrs of incubation in 0.5 mM TBHP, RBC from young donors showed a higher level of hemolysis; instead, RBC from old individuals showed abnormal morphologies, being absent in unstressed RBC, with constriction and budding, which could be identified as poikilocytosis. The same abnormal forms are found in patients with spectrin mutation, leading us to hypothesize that TBHP causes damage to the cytoskeletal spectrin. This suggests that poikilocytosis might be an early stage of red blood cell hemolysis because their presence is associated to a lower level of hemolysis.