Noreddine Ghalim

Beneficial effect on serum apo AI, apo B and Lp AI levels of Ramadan fasting

In order to investigate for the first time in Morocco the effect of fasting in Ramadan, the ninth lunar month of the muslim year, on lipoprotein metabolism, we determined the levels of serum apolipoproteins; apolipoprotein AI (apo AI), apo B, apo AIV and those of lipoprotein particles; apo AI-containing lipoprotein particles (Lp AI) and also apo AI and apo AII containing lipoprotein particles (Lp AI:AII) in a group of 32 healthy, volunteer adult males. Determination of all these parameters was carried out on each week of the month of Ramadan and the results are compared with the pre-fasting and the post-fasting values. Ramadan fasting reduces significantly serum apo B (P < 0.05), while serum apo AI is significantly increased (P < 0.05) compared with the pre-fasting period. The increase of apo AI occurred on day 29 of Ramadan by 11.8%. Serum apo AIV was unchanged during the fasting period indicating that food intake during Ramadan is not based on lipid diet. The observed diet pattern during Ramadan showed an increase of total energy intake based on carbohydrates (+1.4% of total energy), proteins (+0.4% of total energy) but not on fat (-0.7% of total energy), compared with a usual diet used in the rest of the year. The fat diet is high in monounsaturated (P < 0.05) and polyunsaturated fatty acid in contrast to saturated fatty acid which decreased (P < 0.05) during Ramadan. On the other hand, analysis of serum Lp AI and Lp AI:AII showed that the levels of Lp AI:AII were unchanged but those of Lp AI were significantly increased (P < 0.01) at the end of Ramadan. These findings show that feeding behaviour that occurs during Ramadan beneficially affects serum apolipoprotein metabolism and may contribute to prevention of cardiovascular diseases.

Proteomic analysis of Moroccan cobra Naja haje legionis venom using tandem mass spectrometry

UNLABELLED The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. BIOLOGICAL SIGNIFICANCE Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies.

Paraoxonase activity in healthy, diabetic, and hemodialysis patients.

OBJECTIVES Paraoxonase 1 (PON1) is mainly complexed to HDL and is responsible, at least in part, for their antioxidant properties. The aims of our study were to determine the phenotype distribution and enzymatic activities of PON1 and the oxidative stress status of healthy subjects and diabetic and hemodialysis patients. DESIGN AND METHODS PON1 paraoxonase and arylesterase activities and oxidative stress markers [malondialdehyde (MDA) and vitamin E levels] were measured in 300 individuals as a function of health status. RESULTS The prevalence of the PON1 phenotypes in the study population was 74.51%, 18.15% and 7.34% for QQ, QR and RR, respectively. The phenotype distribution did not change significantly as a function of health status (healthy, diabetes, hemodialysis). However, the hemodialysis patients had lower PON1 paraoxonase and arylesterase activities than the diabetic patients and healthy subjects, while there were no significant differences between the diabetic patients and the healthy subjects. Oxidative stress markers (MDA levels and vitamin E/cholesterol ratio) were significantly higher in the diabetic and hemodialysis patients than in the healthy subjects. CONCLUSIONS The lower plasma PON1 enzymatic activities in the hemodialysis patients was not associated with a difference in the phenotype distribution of PON1. Oxidative stress conditions were significantly higher in these patients, which may increase the risk of atherosclerosis in this population.

Comparison between two methods of scorpion venom milking in Morocco

BackgroundThe present study compared two methods used successfully in a large-scale program for the collection of scorpion venoms, namely the milking of adult scorpions via manual and electrical stimulation.ResultsOur immunobiochemical characterizations clearly demonstrate that regularly applied electrical stimulation obtains scorpion venom more easily and, most importantly, in greater quantity. Qualitatively, the electrically collected venom showed lack of hemolymph contaminants such as hemocyanin. In contrast, manual obtainment of venom subjects scorpions to maximal trauma, leading to hemocyanin secretion. Our study highlighted the importance of reducing scorpion trauma during venom milking.ConclusionsIn conclusion, to produce high quality antivenom with specific antibodies, it is necessary to collect venom by the gentler electrical stimulation method.

Snake venomics of Macrovipera mauritanica from Morocco, and assessment of the para-specific immunoreactivity of an experimental monospecific and a commercial antivenoms.

Proteomic analysis of the venom of the medically relevant snake Macrovipera mauritanica from Morocco revealed a complex proteome composed of at least 45 toxins from 9 protein families targeting the hemostatic system of the prey or victim. The toxin profile of Moroccan M. mauritanica displays great similarity, but also worth noting departures, with the previously reported venom proteome of M. lebetina from Tunisia. Despite fine compositional differences between these Macrovipera taxa, their overall venom phenotypes explain the clinical picture observed in M. mauritanica and M. lebetina envenomings. However, M. mauritanica venom also contains significant amounts of orphan molecules whose presence in the venom seems to be difficult to rationalize in the context of a predator-prey arms race. The paraspecific immunoreactivity of an experimental monospecific (M. mauritanica) antivenom and a commercial bivalent antivenom, anti-C. cerastes and anti-M. lebetina, against the venoms of Moroccan M. mauritanica and Tunisian M. lebetina, was also investigated through an affinity chromatography-based antivenomics approach. Both antivenoms very efficiently immunodepleted homologous venom toxins and displayed a high degree of paraspecificity, suggesting the clinical utility of the two antivenoms for treating bites of both M. mauritanica or M. lebetina.

Alteration of HDL functionality and PON1 activities in acute coronary syndrome patients

OBJECTIVE The functionality of HDL has been suggested as an important factor in the prevention of cardiovascular and coronary artery diseases. The objective of the present study was to investigate the functionality of HDL and the factors that may affect the anti-atherogenic properties of HDL in ACS patients. METHODS AND RESULTS One hundred healthy subjects and 205 ACS patients were recruited. HDL functionality was evaluated by measuring their capacity to mediate cholesterol efflux from J774 macrophages. Oxidative stress status was determined by measuring plasma malondialdehyde (MDA), protein carbonyl, and vitamin E levels by HPLC. The PON1 Q192R polymorphism status and PON1 paraoxonase and arylesterase activities of the healthy subjects and ACS patients were also determined. The HDL of ACS patients displayed a limited capacity to mediate cholesterol efflux, especially via the ABCA1-pathway. MDA (7.06±0.29 μM) and protein carbonyl (9.29±0.26 μM) levels were significantly higher in ACS patients than in healthy subjects (2.29±0.21 μM and 3.07±0.17 μM, respectively, p<0.0001), while α- and γ-tocopherol (vitamin E) levels in ACS patients were 8-fold (p<0.001) and 2-fold (p<0.05) lower than in healthy subjects. Paraoxonase, arylesterase and HDL-corrected PON1 activities (PON1 activity/HDL ratio) were significantly lower in ACS patients. Logistic regression analyses showed that high PON1 paraoxonase and arylesterase activities had a significant protective effect (OR=0.413, CI 0.289-0.590, p<0.001; OR=0.232 CI 0.107-0.499, p<0.001, respectively) even when adjusted for HDL level, age, BMI, and PON1 polymorphism. CONCLUSION The results of the present study showed that the functionality of HDL is impaired in ACS patients and that the impairment may be due to oxidative stress and an alteration of PON1 activities.

Serum paraoxonase activity, high-sensitivity C-reactive protein, and lipoprotein disturbances in end-stage renal disease patients on long-term hemodialysis

BACKGROUND Hemodialysis patients are at high risk for atherosclerotic events. Enhanced oxidant stress, dyslipidemia, and inflammation may have a major role in this risk. In this work, we assessed lipoprotein status, total homocysteine, high-sensitivity C-reactive protein (hs-CRP), and paraoxonase activity in hemodialysis patients to determine the correlations among these parameters and to compare these values with those measured in normal control subjects. METHODS We enrolled 109 end-stage renal disease patients on long-term hemodialysis and 100 age- and gender-matched healthy subjects. Total cholesterol, triglycerides, and high-density lipoprotein cholesterol levels were evaluated using colorimetric methods. Low-density lipoprotein (LDL) cholesterol was calculated according to the Friedewald formula. Serum levels of hs-CRP, apolipoproteins (Apo) AI, B, E, and lipoprotein(a) were measured by nephelometry. Lipoprotein particle (Lp) A-I and LpA-I:A-II were determined by immunoelectrophoresis. Total homocysteine levels were evaluated by the fluorescence polarization immunoassay method. Paraoxonase activity was determined using the paraoxon-like substrate. RESULTS Compared with controls, hemodialysis patients had more frequent atherogenic dyslipidemia, hyperhomocysteinemia, and elevated hs-CRP levels. These latter findings inversely correlate with ApoA-I and LpA-I:A-II and positively with ApoB, lipoprotein(a), and ApoB/ApoA-I ratio. Homocysteine levels correlated positively with age. Paraoxonase activity was decreased in hemodialysis patients, especially in elderly patients. This enzyme activity positively correlated with LpA-I:A-II, and inversely with hs-CRP, LDL-cholesterol, and ApoE levels. CONCLUSION The present study demonstrated an abnormal lipoprotein profile associated with increased hs-CRP and decreased paraoxonase activity in hemodialysis patients. Hence, inflammation, dyslipidemia, and increased oxidant stress linked to uremia may be contributors to increased cardiovascular risk in this population.

Association between Paraoxonase 1 (PON1) Polymorphisms and the Risk of Acute Coronary Syndrome in a North African Population.

The purpose of the present study was to investigate the distribution of PON1 Q192R and L55M polymorphisms and activities in a North African population and to determine their association with cardiovascular complications. The prevalence of the QQ, QR, RR, LL, LM, and MM genotypes in the study population was 55.4%, 34.09%, 9.83%, 41.97%, 48.20%, and 9.83% respectively. The Q, R, L, and M alleles had a gene frequency of 0.755, 0.245, 0.67, and 0.33, respectively. The PON1 192 RR genotype was significantly more prevalent among ACS patients than among healthy subjects. There was a 4.33-fold increase in the risk of ACS in subjects presenting the PON1 192 RR genotype compared to those with the QQ genotype (OR=4.33; 95% CI=1.27–17.7). There was a significantly different distribution of PON1 L55M in the ACS patient groups (UA, STEMI, NSTEMI). Moreover, individuals presenting the PON1 55MM genotype present a higher risk for ACS than those with LL genotype (OR=3.69; 95% CI=1.61–11.80). Paraoxonase activities were significantly lower in coronary patients than in healthy subjects. The decrease in PON1 activity was inversely correlated with the number of concomitant risk factors for CVD (r=0.57, p<0.0001). The results of the present study suggested that the PON1 R and M alleles may play a role in the pathogenesis of cardiac ischemia in our North African population and that a decrease in PON1 activity may be a valuable marker for monitoring the development of the atherosclerosis process and the associated cardiovascular complications.

Apolipoprotein AIV of human interstitial fluid is associated with apolipoprotein AI-containing but not with AII-containing particles.

Apolipoproteins and lipoprotein particles from human interstitial fluid and plasma were analyzed. The interstitial fluid was enriched in apolipoproteins AI, AII, and AIV compared with apo B, apo CIII, and apo E. LpAI was found to contain apo AIV which was absent from LpAI: AII. Moreover, the bulk of lecithin-cholesterol acyltransferase was present in LpAI. The concentration range of these particles was in agreement with those required in vitro for cholesterol efflux. Thus the interstitial fluid contains particles in which two agonists but no antagonists of cholesterol efflux are associated with lecithin-cholesterol acyltransferase activity. This supports apolipoprotein AI-and/or AIV-containing particles playing a critical role in the first step of reverse cholesterol transport.

Evaluation of the lethal potency of scorpion and snake venoms and comparison between intraperitoneal and intravenous injection routes.

Scorpion stings and snake bites are major health hazards that lead to suffering of victims and high mortality. Thousands of injuries associated with such stings and bites of venomous animals occur every year worldwide. In North Africa, more than 100,000 scorpion stings and snake bites are reported annually. An appropriate determination of the 50% lethal doses (LD50) of scorpion and snake venoms appears to be an important step to assess (and compare) venom toxic activity. Such LD50 values are also commonly used to evaluate the neutralizing capacity of specific anti-venom batches. In the present work, we determined experimentally the LD50 values of reference scorpion and snake venoms in Swiss mice, and evaluated the influence of two main venom injection routes (i.e., intraperitoneal (IP) versus intravenous (IV)). The analysis of experimental LD50 values obtained with three collected scorpion venoms indicates that Androctonus mauretanicus (Am) is intrinsically more toxic than Androctonus australis hector (Aah) species, whereas the latter is more toxic than Buthus occitanus (Bo). Similar analysis of three representative snake venoms of the Viperidae family shows that Cerastes cerastes (Cc) is more toxic than either Bitis arietans (Ba) or Macrovipera lebetina (Ml) species. Interestingly, the venom of Elapidae cobra snake Naja haje (Nh) is far more toxic than viper venoms Cc, Ml and Ba, in agreement with the known severity of cobra-related envenomation. Also, our data showed that viper venoms are about three-times less toxic when injected IP as compared to IV, distinct from cobra venom Nh which exhibited a similar toxicity when injected IP or IV. Overall, this study clearly highlights the usefulness of procedure standardization, especially regarding the administration route, for evaluating the relative toxicity of individual animal venoms. It also evidenced a marked difference in lethal activity between venoms of cobra and vipers, which, apart from the nature of toxins, might be attributed to the rich composition of high molecular weight enzymes in the case of viper venoms.