General characterization of venom from the Moroccan snakes Macrovipera mauritanica and Cerastes cerastes

Ophidian envenomation accidents constitute a serious public health problem in many countries around the globe. Over 5 million such accident cases occur each year causing more than 100,000 deaths. In Africa, more than 20,000 deaths per year are registered while 400,000 envenomation victims retain severe and permanent functional sequelae. In Morocco, snakebites are frequent and of greater severity in children. They occur mostly in rural areas. The incidence of these bites remains poorly understood and vastly underestimated. The epidemiological data are not well known due to the absence of a national registry, whereas a significant proportion of envenomations receive only traditional treatment methods in non-medical intensive care. This prompted us to investigate the enzymatic and biological properties of venom biochemical constituents from two of the most dangerous snake venoms in Morocco: Cerastes cerastes (Cc) and Macrovipera mauritanica (Mm). Also, we studied the immune cross-reactivity of Cc and Mm venoms in comparison to that of another important dangerous Moroccan viper, Bitis arietans (Ba), to identify the best candidates (venom or a mixture of venoms) for producing the most efficient and protective antivenom. In the present study, we report a preliminary venom characterization of Cc and Mm and the cross-reactivity that may exist between their venoms and Ba. These venoms are known to be highly toxic and contain several proteins that differ by molecular weights. Interestingly, both Cc and Mm venoms are characterized by intense hemorrhagic and phospholipase A2 activities and their ability to degrade the α and γ chains of fibrinogen. They display very low proteolysis through the casein test. After injection into mice, Cc and Mm induce myonecrosis in skeletal muscles, which most likely reflects direct action of myotoxins and indirect action of hemorrhagic molecules present in these venoms. In mice, this myonecrosis diminishes serum creatine phosphokinase (CPK) levels. As expected, Cc venom is immunogenic and induces highly protective antivenom against Mm and Ba venom antigens. This protective capacity is similar to that of the antivenom produced against the Mm venom.(AU)

[Clinical evolution and circulating venom levels in scorpion envenomations in Morocco]. / Evolution clinique et taux circulants du venin dans les envenimations scorpioniques au Maroc.

We conducted a clinical and biological study in Morocco in order to assess the efficacy of antivenom therapy against scorpion stings. Epidemiological and clinical data were collected in 275 patients envenomed by Androctonus mauretanicus mauretanicus and Buthus occitanus scorpions. Patients received antivenom or symptomatic drugs. Blood samples were collected upon hospital admission, at 1 hr and 3 hrs after the treatment. An enzyme linked immunosorbent assay (ELISA) was set up to quantify the venom levels in serum of envenomed patients. Mean serum venom concentrations showed an association between clinical signs and the venom level. The venom concentration at admission, in patients who received 10 ml of antivenom, was significantly reduced after antivenom therapy. The decrease was less important in patients who received only 2 to 5 ml of antivenom. No difference was shown in the venom concentration of patients not treated with antivenom. The clinical signs decreased significantly after antivenom treatment. The absence of antivenom administration increased the risk to develop clinical signs at the end of hospitalisation. This risk was much higher when the delay between scorpion sting and hospital admission increased. The results of our study have demonstrated the efficacy of antivenom in reducing circulating venom and symptoms. Antivenom therapy is more efficient when administered as soon as possible after envenomation and with appropriate quantities of antivenom. This study is favourable to the use of SAS but a prospective study would be useful to confirm these data.

Probucol promotes reverse cholesterol transport in heterozygous familial hypercholesterolemia. Effects on apolipoprotein AI-containing lipoprotein particles.

In order to investigate the effect of Probucol therapy on reverse cholesterol transport, apo AI-containing lipoprotein particles were isolated and characterized, and their cholesterol effluxing capacity and LCAT activity were assayed in four familial hypercholesterolemia patients before and after 12 weeks of Probucol therapy. Four major subpopulations of apo A-containing lipoprotein particles are separated before and after drug treatment; LpAI, LpAI:AII, LpAIV, LpAI:AIV:AII. Probucol reduces both total plasma and LDL-cholesterol (-17 and -14%, respectively). Apo B decreases slightly (-7.6%). Plasma HDL-cholesterol and apo AI decrease by 36.6 and 34.7%. LpA-I showed a marked decrease (-46%). Moreover, plasma LCAT and CETP activities were markedly increased under Probucol treatment. Analysis of lipoprotein particles showed that Probucol induces a decrease of protein content and an increase of cholesterol and triglycerides contents. Interestingly, Probucol induces an enhancement of LCAT activity in LpAI (4.5-fold). This drug induces a trend toward greater cholesterol efflux from cholesterol-preloaded adipose cells promoted by Lp AI and Lp AIV but not by Lp AI:AII. This study confirms the hypothesis, in addition to the lowering LDL-cholesterol levels and antioxidant effects of Probucol, that HDL reduction was not an atherogenic change in HDL system but may cause an antiatherogenic action by accelerating cholesterol transport through HDL system, promoting reverse cholesterol transport from peripheral tissues.

Scorpion envenomation and serotherapy in Morocco.

A clinical and biologic study was conducted in Morocco to assess the efficiency of antivenom therapy for treating victims of scorpion stings. Epidemiologic and clinical data were collected from 275 patients envenomed by Androctonus mauretanicus mauretanicus and Buthus occitanus scorpions. Patients received antivenom or other drugs. Blood samples were collected at the time of hospital admission and 1 hr and 3 hr after treatment. Serum venom levels were quantified by using an ELISA. An association was found between clinical signs of envenoming and the level of venom in serum. Patients classified as grade II (moderate envenoming) had higher serum levels of venom level than patients classified as grade I (mild envenoming). At admission to the hospital, the mean venom concentration was not significantly different between the group not treated with antivenom, the group who received 2-5 ml of antivenom, and the group who received 10 ml of antivenom. A significant decrease in serum venom levels and an improvement in the clinical conditions were observed in patients administered 10 ml of antivenom. The lower decrease in serum venom levels in patients who received 2-5 ml of antivenom was due to lower doses of antivenom. No difference in the venom concentration was observed in patients who were not treated with antivenom. The absence of administration of antivenom increased the risk of developing clinical signs at the end of the hospitalization period. However, this risk was much higher when more than 1 hr elapsed between the time of the scorpion sting and the time of hospital admission. The results demonstrate that antivenom is effective in decreasing circulating venom and morbidity. Serotherapy is more efficient when given as soon as possible after envenomation and with adequate quantities of antivenom.

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.

Fasting during Ramadan induces a marked increase in high-density lipoprotein cholesterol and decrease in low-density lipoprotein cholesterol.

We demonstrated for the first time in a Moroccan population that fasting during Ramadan, the ninth lunar month of the Muslims' year, affected lipid and lipoprotein metabolism in a group of 32 healthy adult male volunteers. This investigation was conducted to study the changes in serum total cholesterol, triglycerides, cholesterol in high-density lipoprotein (HDL) and low-density lipoprotein (LDL), glucose, and body weight during Ramadan. The results showed a significant decrease (7.9%, p < 0.001) in serum total cholesterol concentration during Ramadan as compared with the prefasting period. Also, we obtained a significant decrease of serum triglyceride concentration (30%, p < 0.001) during Ramadan fasting as compared to the period before Ramadan. The reduction of both serum triglycerides and total cholesterol was maintained 1 month after Ramadan. By the end of Ramadan, serum HDL cholesterol had markedly increased (14.3%, p < 0.001) and remained elevated 1 month after Ramadan in contrast to LDL cholesterol which showed a significant decrease (11.7%, p < 0.0001) also maintained 1 month after Ramadan. Mean body weight declined by 2.6% (p < 0.01) on day 29 of Ramadan, whereas during Ramadan, the diet pattern used by our subjects showed an increase of total energy intake due to carbohydrates (+ 1.4% of total energy), proteins (+ 0.4% of total energy) but not fat (-0.7% of total energy) compared to a usual diet used throughout the rest of the year. Moreover, the fat diet is high in monounsaturated (p < 0.05) and polyunsaturated fatty acid in contrast to saturated fatty acid which significantly (p < 0.05) decreased during Ramadan. These findings suggest that feeding behavior that occurs during Ramadan beneficially affects plasma lipids and lipoproteins.

Apolipoproteins and lipoprotein particles in Moroccan patients with previous myocardial infarction.

We investigated for the first time in the Moroccan population the relationship between lipoprotein particles and the progression of coronary atherosclerosis. Plasma lipid variables, including total cholesterol, triglycerides, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, apolipoproteins AI and B, Lp AI, Lp AI: AII, and Lp(a) were measured in 40 Moroccan adults who suffered a verified myocardial infarction before the age of 50 years. The results were compared with a healthy control group. Plasma total cholesterol, triglyceride, and Lp AI: AII levels of patients did not differ significantly from control subjects. Patients had lower plasma high-density lipoprotein-cholesterol (P < 0.05), apo AI (P < 0.05), and Lp AI (P < 0.001) than control subjects, suggesting that the cholesterol reverse transport system is altered in patients with previous myocardial infarction. However, patients had higher plasma low-density lipoprotein-cholesterol (P < 0.001), apo B (P < 0.001), and Lp(a) (P < 0.001). In all patients the best predictor of cardiovascular risk was the independent risk factor Lp(a) plasma level, and the Lp AI plasma level. In this study, the increased coronary atherosclerosis risk with elevated plasma levels of apo B and Lp(a), and with reduced Lp AI, was substantially modified by smoking habits, but not by family history of myocardial infarction.

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 acyl-transferase 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.

Lipoproteins containing apolipoprotein A-IV: composition and relation to cholesterol esterification.

In order to investigate the relationship of lipid and apolipoprotein composition to cholesterol esterification in lipoproteins containing apolipoprotein (apo) A-IV, apo A-containing lipoprotein particles were isolated from fresh human plasma using a system of sequential immunoaffinity chromatography. Plasma was first depleted of apo B- and apo E-containing lipoproteins. Four major subpopulations of apo A-containing lipoprotein particles were separated: Lp A-I, Lp A-I: A-II, Lp A-IV and Lp A-I: A-IV: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained less total lipid, less cholesterol and more triacylglycerol than Lp A-I and Lp A-I: A-II. Lp A-IV and Lp A-I: A-IV: A-II contained more sphingomyelin and less phosphatidylcholine than Lp A-I and Lp A-I: A-II and were richer in (16:0 + 18:0) saturated fatty acids. Among these isolated lipoprotein particles, Lp A-IV contained the highest lecithin: cholesterol acyltransferase (LCAT) activity per micrograms of protein. Cholesterol esterification rates were 2.6 +/- 0.5, 5.3 +/- 0.4 and 0.8 +/- 0.2 mumol of cholesterol per hour per mg of lipoproteins for Lp A-IV, Lp A-I and Lp A-I: A-II, respectively. The apolipoprotein and lipid composition and LCAT activity of Lp A-IV suggest that this lipoprotein may be a source of cholesterol esterification in plasma.

Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid.

Apolipoprotein (apo) A-IV has been proposed to play a role in reverse cholesterol transport. ApoA-IV-containing lipoprotein particles (A-IVLp) were isolated from human plasma and interstitial fluid and characterized by immunoaffinity chromatography. Two major A-IVLp subpopulations, lipoprotein particles containing apoA-IV with apoA-I (LpA-I:A-IV) and lipoprotein particles containing apoA-IV without apoA-I (LpA-IV), were identified. The larger subpopulation of A-IVLp is the LpA-IV that represents 70% (protein mass) of the initial particles. Only 5.8% of apoA-IV was recovered in the retained fraction after affinity chromatography with an anti-apoA-I immunosorbent. ApoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoE, apoH, lecithin: cholesterol acyltransferase (LCAT), cholesteryl ester transfer (CET) protein, proline-rich protein, and a protein of Mr 59,000 were detected in the A-IVLp. These particles contain more than 20% triglycerides (lipid mass). ApoA-IV-containing particles that were isolated from plasma are heterogeneous in size, consisting of two major populations with Stokes' diameters of 10.3 nm and 9.3 nm. Both subpopulations of A-IVLp contain LCAT and CET activities and promote cholesterol efflux from cholesterol-preloaded adipose cells. These data support the hypothesis that A-IVLp particles may be involved in reverse cholesterol transport.

Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells.

Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo AII (LpAI) and particles containing apo AI and apo AII (LpAI:AII) isolated from native human plasma, and from HDL2 or HDL3, showed that only LpAI were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent Kd values between 10 and 25 micrograms/ml). During this long-term exposure, LpAI:AII demonstrated a concentration-dependent inhibition (10-60 micrograms/ml) of LpAI-mediated cholesterol efflux from adipose cells under conditions where LpAI:AII did not deliver cholesterol to the cells and where no net change in the distribution of apo AI between LpAI and LpAI:AII was observed. The antagonizing and modulating role of LpAI:AII in preventing cholesterol efflux mediated by LpAI appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo AI in LpAI particles and apo AII in LpAI:AII particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux.

[Influence of alcohol on plasma lipids and atherogenesis. Epidemiological and biochemical aspects]. / Influence de l'alcool sur les lipides plasmatiques et l'athérogenèse. Aspects épidémiologique et biochimique.

The results of epidemiological and clinical studies published since 1980 concerning the effects of alcohol intake on coronary artery disease are rather contradictory. Although some protective action of alcohol, notably of wine against atherosclerosis, has been described by some authors, the methodological limitations of these studies make it impossible to establish a cause-effect relationship in this matter. Biochemical studies have provided a more precise approach of the effect of alcohol on the mechanism of atherosclerosis. An increase of HDL has been shown in patients who regularly consume alcoholic drinks. However, a detailed analysis of HDL subfractions (and notably HDL2 regarded as an antiatherogenic lipoprotein) has given equally contradictory results. When the antiatherogenic lipoprotein particles present in HDL are accurately identified, the physiopathological consequences of regular alcohol consumption will be more clearly determined. Biochemical and epidemiological information is still insufficient for us to attribute an antiatherogenic effect to alcohol.

Differential electroimmunoassay of human LpA-I lipoprotein particles on ready-to-use plates.

We describe a method for directly measuring LpA-I lipoprotein particles containing apolipoprotein A-I (apo A-I) not associated with apolipoprotein A-II (apo A-II), by differential electroimmunoassay of plasma on ready-to-use plates. Lipoprotein particles containing both apo A-I and apo A-II (LpA-I:A-II) are retained close to the wells when a very high excess of anti-apo A-II is used as compared with anti-apo A-I, whereas the LpA-I particles migrate and react with anti-apo A-I. The method is specific, rapid, and precise. Within- and between-run CVs at three concentrations (high, medium, and low) ranged between 1.51% and 2.72% and 3.01% and 4.56%, respectively. Analytical recovery of isolated LpA-I was from 93% to 115%. Results correlate well with those obtained by two-phase electroimmunoassay, enzyme-linked differential-antibody immunosorbent assay, and immunoaffinity chromatography coupled to enzyme-linked immunosorbent assay. The average normolipidemic concentration of LpA-I was 600 mg/L in 45 women and 490 mg/L in 40 men (P less than 0.0001).

Effect of alcohol intake on human apolipoprotein A-I-containing lipoprotein subfractions.

High-density lipoprotein comprises two main types of lipoprotein particles: (1) those that contain apolipoproteins A-I and A-II, designated LpA-I:A-II, and (2) those that contain apolipoprotein A-I but not apolipoprotein A-II, designated LpA-I. Both have been extensively studied and are believed to represent distinct metabolic entities that may confer differing protection against coronary artery disease risk. We have previously suggested that LpA-I might represent the antiatherogenic effect, which has been ascribed mainly to its effect on high-density lipoprotein cholesterol; we set out to investigate, in 344 men, the relation between LpA-I:A-II and LpA-I levels and alcohol consumption. As the alcohol intake rose, LpA-I:A-II levels increased, while LpA-I levels fell. On the assumption that LpA-I is the antiatherogenic fraction of high-density lipoprotein, the putative protective action of alcohol consumption against coronary artery disease should be reconsidered.

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells.

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.

[Quantitative analysis of apolipoproteins and lipoprotein particles in patients with head injuries]. / Analyse quantitative des apolipoprotéines et des particules lipoprotéiniques chez les traumatisés crâniens.

By head-injured patients, apo A-I and apo A-II concentrations were more decreased in HDL3 than in HDL2. Then, the plasmatic concentrations of the main lipoprotein particles present in HDL fraction were modified. For example, a significant decrease of Lp A-I: A-II particles was observed and this variation was similar to that of total apo A-I (r = 0.78). On the other hand, the concentration of Lp A-I particles was slightly modified, apo C-III concentration was markedly decreased whereas apo E concentration was significantly increased (p less than 0.05); in plasma samples obtained 10 days after a severe head injury, apo E reached three times the normal value.