Protective effects of selected medicinal plants against protein degradation, lipid peroxidation and deformability loss of oxidatively stressed human erythrocytes.

The effects of seven medicinal plants including Artemisia herba-alba, Ferula hermonis, Hibiscus sabdariffa, Nigella sativa, Teucrium polium, Trigonella foenum-graecum, and Allium sativum on protein degradation, lipid peroxidation, erythrocyte deformability and osmotic fragility of erythrocytes exposed in vitro to 10 mM H(2)O(2) for 60 min at 37 degrees C have been examined. Preincubation of erythrocytes with Nigella sativa and Allium sativum protected erythrocytes against protein degradation, loss of deformability and increased osmotic fragility caused by H(2)O(2), while the other plants failed to protect erythrocytes against these damages. Artemisia herba-alba did not protect erythrocytes against lipid peroxidation, while Trigonella foenum-graecum unexpectedly increased lipid peroxidation of erythrocytes exposed to H(2)O(2). Ferula hermonis, Hibiscus sabdariffa, Nigella sativa, Teucrium polium and Allium sativum protected erythrocytes against lipid peroxidation. The results indicate the importance of oxidatively damaged cellular proteins in compromising the rheologic behaviour of the erythrocytes, and that the medicinal plants which have anti-protein-oxidant activity (e.g. Nigella sativa and Allium sativum) could be rheologically useful, particularly in pathological conditions related to free radicals.

Exposure of human neutrophils to oxygen radicals causes loss of deformability, lipid peroxidation, protein degradation, respiratory burst activation and loss of migration.

The effects of oxygen radical generating system of H(2)O(2) on neutrophil deformability, lipid peroxidation, protein degradation, chemotaxis, random migration and activation were studied in the presence and absence of selected anti-oxidants such as vitamin E and carbon monoxide. Hydrogen peroxide caused a significant loss of neutrophil deformability (measured as an index of filtration), loss of chemotaxis (using chemoattractant) and loss of random migration (spontaneous migration) under agarose gel. Hydrogen peroxide also caused a significant increase in lipid peroxidation (measured as malonyldialdehye) and protein degradation (measured as free alanine). Hydrogen peroxide also caused activation of neutrophils (measured by nitroblue tetrazolium dye test). Pre-incubation of neutrophils with vitamin E prevented significantly the increase in lipid peroxidation without affecting the loss of neutrophil deformability. However, pre-incubation with carbon monoxide prevented significantly the increase in protein degradation and the loss of neutrophil deformability. The results indicate the importance of oxidatively damaged cell proteins in compromising the rheologic behaviour and migration of neutrophils, particularly under oxidative stress conditions.

Susceptibility of erythrocytes from non-insulin-dependent diabetes mellitus and hemodialysis patients, cigarette smokers and normal subjects to in vitro oxidative stress and loss of deformability.

Incubation of normal erythrocytes with 10 mM H2O2 has caused a loss of deformability. This loss of deformability was correlated with the extent of malonyldialdehyde, MDA (an indicator of lipid peroxidation) and alanine production (an indicator of protein degradation). The susceptibility of erythrocytes from 21 non-insulin-dependent diabetes mellitus (NIDDM) and 18 hemodialysis patients, 21 cigarette smokers and 25 healthy controls to in vitro oxidative stress with H2O2 has been measured as MDA production. Besides this, their erythrocytes reduced glutathione (GSH; an antioxidant) level has also been determined, but before exposure to H2O2. Erythrocytes from NIDDM and hemodialysis patients have shown significant increase in MDA production and a significantly low GSH level, compared to healthy controls. In cigarette smokers, although the GSH level was significantly low, but there was no significant difference in MDA production, compared to healthy controls. The low GSH level in NIDDM and hemodialysis patients, and smokers indicates that their erythrocytes were exposed to oxidative stress (an increase in free radical load) in vivo, resulting in an overconsumption and/or decreased production of GSH. The increased susceptibility to oxidative stress along with the decrease in some antioxidants (e.g., GSH) may explain the significant increase in MDA production in NIDDM and hemodialysis patients. But in cigarette smokers, the increased susceptibility to oxidative stress is probably not sufficient to cause a significant increase in MDA production. The results may also indicate an increased susceptibility to the loss of erythrocyte deformability in NIDDM and hemodialysis patients compared to healthy controls.

Exposure of human erythrocytes to oxygen radicals causes loss of deformability, increased osmotic fragility, lipid peroxidation and protein degradation.

The effects of two oxygen radical generating systems (H2O2 and ascorbate/Fe+2) on erythrocyte deformability, osmotic fragility, lipid peroxidation and protein degradation were studied. Incubation of erythrocytes with different concentrations of H202 (5-20 mM) or ascorbate/Fe+2 (10/0.1-40/0.4 mM) caused a loss of deformability and increased osmotic fragility. The loss of deformability has occurred in a dose-dependent fashion and was proportional to the extent of malonyldialdehyde (an indicator of lipid peroxidation) and alanine production (an indicator of protein degradation). Prior exposure of the erythrocytes to carbon monoxide (known to inhibit heme-protein degradation) prevented almost completely the loss in deformability caused by H2O2, indicating that the loss in deformability was due mainly to protein degradation rather than to lipid peroxidation. Erythrocytes incubated with either of the two systems have also shown morphologic changes characterized by a dose-dependent increase in echinocyte formation. The results indicate the importance of oxidatively damaged proteins in compromising the rheologic behaviour of the erythrocytes, particularly when the free radicals are involved.

Evaluation of different methods used to measure malonyldialdehyde in human erythrocytes.

Malonyldialdehyde (MDA), a secondary product of lipid peroxidation is widely used as an indicator of lipid peroxidation. Eight of the most frequently used methods for measuring MDA have been evaluated with regard to their sensitivity and reproducibility. The sensitivity of these methods for pure MDA solution was in the order: Satoh's > Stocks and Dormandy's >> Buege and Aust's > Dresel's >> Slater's > Yoshioka's et al. > Yagi's > Jain's method. Whereas the sensitivity of the first four methods for erythrocyte MDA was in the order: Stocks and Dormandy's >> Buege and Aust's > Satoh's > Dresel's method. The reproducibility (expressed as coefficient of variation) of these four methods for erythrocyte MDA were: 3.5%, 17.9%, 31.5% and 16.1%, respectively. These results indicate that Stocks and Dormandy's method has the highest sensitivity and an excellent reproducibility for erythrocyte MDA. Also, it was found to be simple and many samples can be treated in a relatively short time. When standard MDA (0.1-15 nmol/ml) was incubated with erythrocytes, the percentage recovery of MDA (using Stocks and Dormandy's method) has ranged from 50-85%. This result indicates that a considerable amount of MDA formed in erythrocytes probably reacts with other cell components and becomes undetectable. Despite this, the determination of MDA level remains a useful indicator of lipid peroxidation and correlates well with the degree of oxidative stress.

Rheological action of aspirin on human erythrocytes.

The rheological action of aspirin on human erythrocytes was studied by in vitro incubation of normal erythrocytes with aspirin at 1, 2 and 4 mg/ml for 30-60 min and then measuring erythrocyte rheological properties of deformability, osmotic fragility and aggregation. Aspirin (2 and/or 4 mg/ml) significantly (p<0.05) prevented the loss of filterability (deformability) through 5 microm diameter pores of erythrocytes dehydrated with hypertonic buffer (450 mOsm/kg water) or with potassium ionophore valinomycin (18 micromol/l). When the calcium ionophore A23187 (1.9 micromol/l) was used to induce cell dehydration, aspirin (4 mg/ml) unexpectedly significantly (p<0.05) increased further the loss of filterability (deformability). Aspirin (1, 2 and 4 mg/ml) also increased significantly (p<0.05) erythrocyte osmotic fragility. Aspirin (4 mg/ml) had no effects on aggregation of erythrocytes induced by dextran 70 (3%). The results suggest that aspirin could play a rheologically active role on erythrocytes. The observed effects of aspirin could be explained by acetylation of intracellular proteins and hence saturation (concentration) of the cell interior with the osmotically active drug.

Consensus and accuracy in haematology laboratories of developing countries: the Jordanian experience.

A study lasting for 18 months using interlaboratory surveys was carried out to assess the analytical quality of Jordanian haematology laboratories that represent one of the developing countries. The study surveyed 50 laboratories constituting the majority of clinical laboratories in the central region of Jordan using 15 control specimens of whole fresh blood and eight freshly prepared blood smears. The study covered the routine haematological parameters of PCV, Hb, RBC, WBC and differential white blood cell count. More than 97% of Jordanian laboratories using cell counters achieved the medically useful criteria for analytical performance, this figure was reduced to 84% in laboratories using manual methods. Jordanian laboratories, however, were far from achieving the analytical goals that have been proposed based on intraindividual biological variation. This study stressed the need for a national EQA scheme in haematology, to reach a common level of standardization.

Screening secondary school students in the Gaza strip for beta-thalassaemia trait.

A small scale screening study for beta-thalassaemia trait has been carried out in the Gaza Strip, involving 1650 secondary schools healthy students, 16-18 years old and from both sexes. The results showed that the overall prevalence in the Gaza Strip of beta-thalassaemia was 4.3%. The frequency of beta-thalassaemia trait in the microcytic (defined as MCV < or = 80fl and/or MCH < or = 26 pg) subjects was 27.1%. The efficacies of some of the proposed discrimination functions in the differentiation between beta-thalassaemia trait and non-thalassemic microcytosis were evaluated. The Mentzer index, MCV of < or = 72fl, England & Fraser DF and the Shine & Lal formula were found to correctly identify 91.6%, 82.4%, 81.3% and 62.6% of the studied cases of microcytosis as having or not having the beta-thalassaemia trait. It was concluded that both beta-thalassaemia and microcytic anaemias are major health problems in the Gaza Strip. The various forms of consanguineous marriages, in addition to poor economic conditions in the Gaza Strip may have contributed to the concentration of beta-thalassaemia and the prevalence of microcytic anaemias in this population.

Effects of selected flavonoids on deformability, osmotic fragility and aggregation of human erythrocytes.

The effects of 8 selected flavonoids of deformability, osmotic fragility and dextran-induced aggregation of human erythrocytes were studied. The following flavonoids were found to protect against loss of filterability (deformability) through 5 microns diameter pores of erythrocytes dehydrated with calcium ionophore A23187 (1.9 mumol/l): apigenin > quercetin > cirsimaritin > rutin > luteolin > chrysoeriol-4'-O-glucoside > 3,5,7-trihydroxy 4'-methoxy flavone 7-rutinoside, whereas beta-naphtho flavone enhanced the loss of filterability. When the potassium ionophore valinomycin (18 mumol/l) was used to induce cell dehydration, the order of potency of the flavonoids in protecting against loss of filterability was apigenin > cirsimaritin = chrysoeriol-4'-O-glucoside > 3,5,7-trihydroxy 4'-methoxy flavone 7-rutinoside > luteolin = rutin > quercetin, whereas beta-naphtho flavone again enhanced the loss of filterability. All flavonoids reduced ESR measured over 1 h except for 3,5,7-trihydroxy 4'-methoxy flavone 7-rutinoside which showed no effect, and for quercetin which significantly enhanced ESR. All flavonoids also improved erythrocyte osmotic fragility except for apigenin which significantly increased osmotic fragility. These effects were explained in terms of the number and location of hydroxyl groups on the basic skeleton of flavone. The results suggest that the presence of an OH and C5 is essential for the described rheological effects of these flavonoids.

Residues of chlorinated hydrocarbon insecticides in human milk of Jordanian women.

A total of 411 milk samples were screened for the residues of the following chlorinated hydrocarbon insecticides using chromatographic methods: DDT insecticides (P,P'-DDT, P,P'-DDE, P,P'-DDD, O,P' -DDT); cyclodiene insecticides (aldrin, endrin, dieldrin, heptachlor, heptachlorepoxide, cis-and trans-chlordane); hexachlorocyclohexane (alpha-HCH, gamma-HCH) and hexachlorobenzene (HCB). The milk samples were obtained from lactating women selected randomly from five different geographical regions in Jordan. These regions were North and Middle Jordan Valley, Amman, Irbid and Zarqa. All of the studied insecticides were found to contaminate human milk in Jordan, but with high variations in the concentration and percentage of population occurrence. P,P'-DDE was the predominant contaminant occurring in 80.3% of studied population, followed by P,P'-DDT occurring in 53.5%. The regional results showed that the DDT residues were also the predominant contaminant in all regions and that the North and Middle Jordan Valley regions were the most affected. Infant's age (i.e. lactation period) had a significant inverse relationship with the concentration of insecticides in the milk fat.

External quality assessment of Jordanian clinical chemistry laboratories.

A two-year study using inter-laboratory surveys has been carried out to assess the analytical quality of Jordanian clinical chemistry laboratories. The study surveyed greater than 65% (60 laboratories) of Jordanian laboratories using 18 control specimens and covering a total of 15 analytes. Close agreement was obtained between the consensus values and the designated values for analytes which had mean values within the normal range, whereas significantly lower consensus values were obtained for glucose, creatinine, bilirubin and urea in the pathological range. Considerable interlaboratory variation was observed in Jordan relative to EQA schemes in other countries. This study highlighted several problems in Jordanian laboratories, and stressed the need for a national EQA scheme with an effective means of providing continuous advice, education and training in clinical chemistry.

Rheological action of drugs that prevent erythrocyte dehydration.

The water content of the human erythrocyte is a major determinant of its cytoplasmic viscosity and thus deformability. Loss of cell water may be either primary or secondary to loss of erythrocyte cations (K+). Several existing drugs (cetiedil citrate, pentoxifylline and piracetam) have recently been shown to inhibit K+ loss from erythrocytes and thus have the potential to prevent erythrocyte dehydration. Further studies of cation flux pathways in the erythrocyte membrane are of importance for the development of new drugs that maintain cell hydration.

Oxpentifylline and cetiedil citrate improve deformability of dehydrated sickle cells.

Erythrocytes from 14 patients with homozygous sickle cell anaemia were treated with the calcium ionophore A23187 to induce loss of cellular potassium and water. The dehydrated cells showed a decrease in filterability (loss of deformability) through pores of 5 micron diameter. Oxpentifylline and cetiedil citrate, which preserve erythrocyte cation and water content, had a significant (p less than 0.01) protective effect against loss of deformability at a concentration of 1 mumol/l. Oxpentifylline showed no adverse effect on the rheology, morphology, or haemolysis of sickle cells at concentrations up to 500 mumol/l. Drugs that act on the erythrocyte membrane to maintain cell hydration are of potential rheological benefit in sickle cell anaemia.