[Evaluation of the ExSel® self-questionnaire to screen for an excess salt intake in patients followed in a nephrology consultation]. / Évaluation de l'autoquestionnaire ExSel® pour dépister une consommation excessive de sel chez les patients consultant en néphrologie.

AIM: To evaluate the reliability of the ExSel® self-questionnaire to detect an excess salt intake (≥12g/24h) in patients consulting for hypertension and/or renal failure. METHODS: Results of the ExSel® self-questionnaire were compared to 24h sodium excretion using the Cohen's kappa test and a Chi2 test. Sensitivity, specificity, VPP and VPN were calculated. A ROC curve was realized to find an accurate cut-off. RESULTS: Mean characteristics of the 101 patients with reliable results were: age of 67±12 years, Body Mass Index 28.4±5.6kg/m2, SBP/DBP 139±23/74±13mmHg (98% were hypertensives. Mean salt intake was 7.5±3.1g/24h and mean creatininuria was 13.9±20.1mmol/24h. An excess salt intake (≥12g/24h) was observed in 8% of the patients. The Kappa test at 0.17 and the Chi2 at 0.66 signify that the agreement was very low. Sensitivity was 37%, specificity 90%, PPV 20% and NPV 94%. The AUC under the ROC curve was too low (0.665) to determine a threshold adapted to the renal patients. CONCLUSIONS: The ExSel® autoquestionnaire is not adapted to outpatients, mainly hypertensives (98%) followed in a nephrology consultation to detect an excess salt consumption.

[Microalbuminuria and urinary albumin excretion: French guidelines]. / Microalbuminurie et excrétion urinaire d'albumine: recommandations pour la pratique clinique.

UNLABELLED: Measurement of urinary albumin excretion (UAE) may be done on a morning urinary sample or on a 24 hour-urine sample. Values defining microalbuminuria are: - 24-hour urine sample: 30-300 mg/24 hours - Morning urine sample: 20-200 mg/mL or 30-300 mg/g creatinine or 2.5-25 mg/mmol creatinine (men) or 3.5-35 mg/mol (women). - Timed urine sample: 20-200 mug/min. The optimal use of semi-quantitative urine test-strip is not clearly defined. It is generally believed that microalbuminuria reflects a generalized impairment of the endothelium; however, no definite proof has been shown in humans. In diabetic subjects, microalbuminuria is a marker of increased risk of cardiovascular (CV) and renal morbidity and mortality in type 1 and type 2 diabetic subjects. The increase in UAE during follow-up is also a marker of CV and renal risk in type 1 and type 2 diabetic subjects; its decrease during follow-up is associated with lower risks. In non-diabetic subjects, microalbuminuria is a marker of increased risk for diabetes mellitus, deterioration of the renal function, CV morbidity and all-cause mortality. It is a marker of increased risk for the development of hypertension in normotensive subjects, and is associated with unfavorable outcome in patients with cancer and lymphoma. Persistence or elevation of UAE overtime is associated with deleterious outcome in some hypertensive subjects. Measurement of UAE may be recommended in hypertensive subjects with 1 or 2 CV risk factors in whom CV risk remains difficult to assess, and in those with refractory hypertension: microalbuminuria indicates a high CV risk and must lead to strict control of arterial pressure. Studies focused on microalbuminuria in non-diabetic, non-hypertensive subjects are limited; most of them suggest that microalbuminuria predicts CV complications and deleterious outcome as it is in diabetic or hypertensive subjects. Subjects with a history of CV or cerebrovascular disease have an even greater CV risk if microalbuminuria is present than if it is not; however, in all cases, therapeutic intervention must be aggressive regardless of whether microalbuminuria is present or not. It is not recommended to measure UAE in non-diabetic non-hypertensive subjects in the absence of history of renal disease. Monitoring of renal function (UAE, serum creatinine and estimation of GFR) is annually recommended in all subjects with microalbuminuria. MANAGEMENT: in patients with microalbuminuria, weight reduction, sodium restriction (< 6 g/day), smoking cessation, strict glucose control in diabetic subjects, strict arterial pressure control are necessary; in diabetic subjects: use of maximal doses of ACEI or ARB are recommended; ACEI/ARB and thiazides have synergistic actions on arterial pressure and reduction of UAE; in non-diabetic subjects, any of the five classes of anti-hypertensive medications (ACEI, ARB, thiazides, calcium channel blockers or beta-blockers) can be used.

The effect of prolonged administration of ethanol on cardiac metabolism and performance in the dog.

The effect of prolonged administration of alcohol on mitochondrial function and high-energy phosphate (ATP) of heart muscle was investigated in dogs. Animals were divided into two groups, a control group and a group that received alcohol. In the experimental series, dogs received 400 ml of a 25% solution of alcohol added to the food and drinking water. Measurements were carried out after ethanol had been withheld for 2 days. Total myocardial blood flow, cardiac output, and myocardial O(2) consumption remained at control levels. Measurement of cardiac contractility using the maximal rate of left ventricular pressure rise (dP/dt(max)) showed no change in animals exposed to alcohol. When the afterload of the heart was increased with angiotensin, a slight but not significant decline in cardiac contractility was observed. Activities of various intramitochondrial and extramitochondrial enzymes were measured in both groups. After alcohol administration, the primarily intramitochondrial isocitrate dehydrogenase diminished. ATP in heart muscle of dogs exposed to alcohol declined, and mitochondrial oxygen consumption and respiratory control indices diminished. These observations suggest that the primary lesion leading to alteration of myocardial performance is a biochemical malfunction of the mitochondria, which at this early stage is not reflected in changes in myocardial contractility.

Insulin, C-peptide and proinsulin for the biochemical diagnosis of hypoglycaemia related to endogenous hyperinsulinism.

OBJECTIVE: We evaluated the respective value of insulin, C-peptide and proinsulin levels in 33 patients with endogenous hyperinsulinism and in 67 controls to determine the best parameters and thresholds to make or to rule out the diagnosis of endogenous hyperinsulinism. RESULTS: When blood glucose levels were below 2.5 mmol/l, insulin was <21 pmol/l in 8-35% of the patients and in all controls; C-peptide was >0.2 nmol/l in all insulinomas but not in the nesidioblastosis or in the controls; proinsulin was >5 pmol/l in all patients but not in the controls. When fasting blood glucose levels reached 2.5-3.3 mmol/l, proinsulin was <22 pmol/l in all the controls and >22 pmol/l in 74% of the patients. Proinsulin after an overnight fast was below 22 pmol/l in all non-obese controls and above 22 pmol/l in 73% of non-obese patients. CONCLUSION: Proinsulin levels above 5 pmol/l with blood glucose levels below 2.5 mmol/l during a 72 h fast test represent the best criterion for the diagnosis of endogenous hyperinsulinism, reaching 100% diagnostic specificity and sensitivity. Concomitant C-peptide levels above 0.2 nmol/l also make the diagnosis of all our insulinoma patients, not the diagnosis of nesidioblastosis, while insulin levels have much less diagnostic accuracy. Whether proinsulin levels above 22 pmol/l could also make the diagnosis of endogenous hyperinsulinism in part of the patients at the time of fasting blood glucose levels between 2.5 and 3.3 mmol/l or after an overnight fast in non-obese subjects needs further study.

Microalbuminuria and urinary albumin excretion: French clinical practice guidelines.

Urinary albumin excretion (UAE) may be assayed on a morning urinary sample or a 24 h-urine sample. Values defining microalbuminuria are: 1) 24-h urine sample: 30-300 mg/24 h; 2) morning urine sample: 20-200 mg/ml or 30-300 mg/g creatinine or 2.5-25 mg/mmol creatinine (men) or 3.5-35 mg/mmol (women); 3) timed urine sample: 20-200 mug/min. The optimal use of semi-quantitative urine test-strip is not clearly defined. It is generally believed that microalbuminuria reflects a generalized impairment of the endothelium; however, no definite proof has been obtained in humans. IN DIABETIC SUBJECTS: Microalbuminuria is a marker of increased risk of cardiovascular (CV) and renal morbidity and mortality in type 1 and type 2 diabetic subjects. The increase in UAE during follow-up is associated with greater CV and renal risks in type 1 and type 2 diabetic subjects; its decrease during follow-up is associated with lower risks. IN NON-DIABETIC SUBJECTS: Microalbuminuria is a marker of increased risk for diabetes mellitus, deterioration of renal function, CV morbidity and all-cause mortality. It is a marker of increased risk for the development of hypertension in normotensive subjects, and is associated with unfavorable outcome in patients with cancer and lymphoma. Persistence of elevated UAE during follow-up is associated with poor outcome in some hypertensive subjects. Measurement of UAE may be recommended in hypertensive medium-risk subjects with 1 or 2 CV risk factors in whom CV risk remains difficult to assess, and in those with refractory hypertension: microalbuminuria indicates a high CV risk and must lead to strict control of arterial pressure. Studies focused on microalbuminuria in non-diabetic non-hypertensive subjects are limited; most of them suggest that microalbuminuria predicts CV complications and deleterious outcome. Subjects with a history of CV or cerebrovascular disease have an even greater CV risk if microalbuminuria is present than if it is not; however, in all cases, therapeutic intervention must be aggressive regardless of whether microalbuminuria is present or not. It is not recommended to measure UAE in non-diabetic non-hypertensive subjects in the absence of history of renal disease. Monitoring of renal function (UAE, serum creatinine and estimation of GFR) is recommended annually in all subjects with microalbuminuria. MANAGEMENT: In patients with microalbuminuria, weight reduction, sodium restriction (<6 g per day), smoking cessation, strict glucose control in diabetic subjects, strict arterial pressure control are necessary; in diabetic subjects: use of maximal doses of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) are recommended; ACEI/ARB and thiazides have synergistic actions on arterial pressure and reduction of UAE; in non-diabetic subjects, any of the five classes of anti-hypertensive medications (ACEI, ARB, thiazides, calcium channel blockers or beta-blockers) can be used.

[Management of hypertension and obesity: the observatory of management of uncontrolled hypertensives with respect to the presence or absence of overweight (PHYSIObs)]. / Prise en charge de l'hypertension artérielle et surcharge pondérale Observatoire de la prise en charge de patients hypertendus non contrôlés en fonction de la présence ou non d'un surpoids (PHYSIObs).

The object of this study was to compare the management of uncontrolled hypertensives (BP > 140/90 mmHg) by general practitioners with respect to the presence or absence of overweight (BMI > or =25 Kg/m2). A 2/1 stratification allowed comparison of 4080 patients who were overweight and 1951 patients with a normal body weight (normal BMI < 25 Kg/m2). The BP of patients who were overweight (> or =25 Kg/m2) was slightly higher than those with a normal BMI (161 +/- 12 mmHg vs. 159 +/- 12 mmHg, p < 0.001). The presence of a metabolic syndrome (43% vs. 7%, ATPIII criteria) was, logically, commoner in the patients overweight. However, the practitioners only recognised the presence of a metabolic syndrome in 65% of the overweight patients (28% true positives and 37% true negatives). The practitioners fixed their target value of systolic BP at 136.5 +/- 5.6 mmHg, in accordance with the recent recommendations of the Health Authorities. The targets were judged to be difficult to obtain in 18% of the overweight group and in 5% of patients with normal body weights. This optimism contrasted with the prescriptions, especially in the overweight patients, 46% of whom were treated by monotherapy and who remained for 44% on monotherapy at the end of the consultation. This descriptive study confirms the lack of awareness of the metabolic syndrome in overweight patients and identifies barriers to effective management of the hypertension of these high risk patients.

[Home blood pressure monitoring in addition to office blood pressure determination is useful in patients with systolic hypertension before their inclusion into a drug trial]. / L'automesure tensionnelle en complément de la mesure de consultation est utile pour l'inclusion dans un essai clinique de sujets avec une HTA systolique.

OBJECTIVE: In patients with uncontrolled systolic hypertension, to estimate the value of home blood pressure monitoring in addition to office blood pressure for inclusion in a trial. METHODS: 80 patients with systolic hypertension, defined as SBP > or =140 mmHg and pulse pressure > or =60 mmHg, were treated for 4 weeks with a thiazide diuretic at usual dose (25 mg HCTZ or 1.5 mg indapamide or methyclothiazide 5 mg). Blood pressure was measured using an automatic monitor (Omron M6) at office and at home in the 3 days prior the visit. Subjects with an uncontrolled hypertension were included in the second part of the trial only if there fulfilled inclusion criteria: office SBP > or =140 mmHg and home SBP > or =135 mmHg (mean of 18 measurements obtained on 3 consecutive days) and office pulse pressure > or =60 mmHg. RESULTS: After 4 weeks with diuretic treatment, 62% of patients fulfilled 3 criteria and were included in the second part of the trial. It was observed 76% of patients with office SBP > or =140 mmHg, 72% with office pulse pressure > or =60 mmHg and 70% with both office SBP and PP criteria. However, only 67% of patients had home SBP > or =135 mmHg. Discrepancy between office and home SBP was observed and subjects with a white coat hypertension was noticed in 14% and masked hypertension in 5%. CONCLUSION: If patients with systolic hypertension have to be included into a drug trial because there are uncontrolled, home blood pressure monitoring in addition to office blood pressure is a very useful criteria for inclusion because misclassifications due to white coat or masked hypertension is frequent in these patients.

[Percutaneous closure of three septal defects by two Amplatz occluders implanted during the same procedure]. / Fermeture percutanée de 3 communications interauriculaires par 2 dispositifs d'Amplatz implantés au cours d'un même cathétérisme.

Multiple atrial septal defects can be closed by interventional catheterisation. The procedure requires an accurate morphological evaluation: number of defects, distance from their edges to the main cardiac structures, resistance of the septum. The authors report the case of a 63 year old woman presenting with cardiac failure in whom 3 atrial septal defects were diagnosed. All 3 defects were successfully closed by the implantation of two Amplatz devices. Control echocardiography at 6 months showed the occluders in a normal position with no residual shunt and the patient was asymptomatic.

[Myocardial infarction in a young female smoker taking oral contraception]. / Infarctus du myocarde chez une jeune femme fumeuse sous contraception orale.

A 33 year old woman suffered a lateral myocardial infarction for the first time, and was treated by pre-hospital thrombolysis and secondary angioplasty on the diagonal artery. Fifteen days before the cardiac event she had undergone a left ovarian cyst excision and left salpingectomy for an ectopic pregnancy. She was a moderate smoker and had been taking a second-generation biphasic minidose oral contraceptive (ethinyl-estradiol 30-40mg and levonorgestrel 150-200 mg) for about ten years. Fifteen days before the myocardial infarction and due to the ectopic pregnancy she had changed to a combined monophasic minidose oral contraceptive pill containing ethinylestradiol (30 mg) and drospirenone (3 mg). The eventual outcome was favourable, with no complications. In this article we discuss the possible implications of the various factors (oral contraceptive, tobacco use, and surgical intervention) in this young woman with a myocardial infarction.

[Cardiac asystole during acute anterior myocardial infarction: a consequence of endocardiac reflexes]. / Asystolie en phase aiguë d'infarctus du myocarde antéroseptal: une manifestation de la mise en jeu de réflexes endocardiaques.

The authors report a case of paroxysmal, complete atrioventricular block during an anterior acute myocardial infarction, leading to asystolia. The different possible physiopathological mechanisms are discussed, suggesting a paroxysmal nodal conduction defect, secondary to transient parasympathetic stimulation, triggered by a Bezold-Jarish type of cardiac reflex. This reflex is frequently involved in various pathologic situations or diagnostic procedures, usual in cardiology. Although it is frequently observed in inferior myocardial infarction, it can occur during an anterior acute myocardial infarction.

Management of resistant hypertension: expert consensus statement from the French Society of Hypertension, an affiliate of the French Society of Cardiology.

To improve the management of resistant hypertension, the French Society of Hypertension, an affiliate of the French Society of Cardiology, has published a set of eleven recommendations. The primary objective is to provide the most up-to-date information based on the strongest scientific rationale and that is easily applicable to daily clinical practice. Resistant hypertension is defined as uncontrolled blood pressure on office measurements and confirmed by out-of-office measurements despite a therapeutic strategy comprising appropriate lifestyle and dietary measures and the concurrent use of three antihypertensive agents including a thiazide diuretic, a renin-angiotensin system blocker (ARB or ACEI) and a calcium channel blocker, for at least 4 weeks, at optimal doses. Treatment compliance must be closely monitored, as must factors that are likely to affect treatment resistance (excessive dietary salt intake, alcohol, depression, drug interactions and vasopressor drugs). If the diagnosis of resistant hypertension is confirmed, the patient should be referred to a hypertension specialist to screen for potential target organ damage and secondary causes of hypertension. The recommended treatment regimen is a combination therapy comprising four treatment classes, including spironolactone (12.5-25 mg per day). In the event of a contraindication or a non-response to spironolactone, or if adverse effects occur, a ß-blocker, an α-blocker, or a centrally acting antihypertensive drug should be prescribed. Because renal denervation is still undergoing assessment for the treatment of hypertension, this technique should only be prescribed by a specialist hypertension clinic.

Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. I. Action on carboxyl esters, glycerides and phospholipids.

Purified carboxyl ester hydrolase (carboxylic-ester hydrolase, EC 3.1.1.1) from human pancreatic juice was found to hydrolyze triacetin, methyl butyrate and glycerides solubilized by bile salts. It has no activity on substrate presented as emulsoin or monomolecular films. The human enzyme was found to deacylate phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phosphatidylcholines was dependent of substrate solubility and dioctanoyl phosphatidylcholine was deacylated with the highest rate. Long-chain phosphatidylcholines and lysophosphatidylcholines present in microsomal membranes were deacylated with very low rates, only lysophosphatidylcholine deacylation was faster. Evidence is presented that human carboxyl ester hydrolase is the lyophosphatidyl-choline-hydrolyzing enzyme corresponding to bovine lysophospholipase. Bile salts play an important part on the activity of human carboxyl ester hydrolase, in addition to the role of detergent that they have on insoluble substrates.

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients.

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. I. Studies on neutral glycerides.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9-10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein greater than 1(3)-monoolein greater than tributyrin = triacetin greater than or equal to triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[3H]palmitoyl-2-[14C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[14C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. II. Studies on glycerophospholipids.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various natural and synthetic phospholipids. Natural glycerophospholipids carrying a 1-acyl-bond were degraded in the following order of decreasing activity: phosphatidylcholine = phosphatidylinositol greater than 1-acyl-sn-glycero-3-phosphocholine greater than phosphatidylethanolamine greater than phosphatidylglycerol. Sodium deoxycholate was an activator with all the phospholipids tested, each one requiring its own optimal concentration of detergent. Whereas 1-alkyl-2-acyl-sn-glycero-3-phosphocholine remained fully insensitive to enzyme degradation, 2-acyl-sn-glycero-3-phosphocholine was hydrolysed to some extent. However, additional experiments involving time-course hydrolysis revealed that this was entirely due to the migration of the 2-acyl-chain to the sn-1 position. From studies using racemic or enantiomeric phosphatidylcholines, it was concluded that the enzymes are not stereospecific. Activity against 1-acylpropanediolphosphocholine was much lower than with 1-acyl-sn-glycero-3-phosphocholine, indicating that the 2-hydroxyl group (or the 2-acyl-ester group) participates in the substrate reactivity through a strong inductive effect. Some activity could be detected against 1,3-diacylglycero-2-phosphocholine (beta-phosphatidylcholine) and 1-acylglycol-2-phosphocholine. It is thus concluded that the failure of the lipases to hydrolyse the 2-acyl-bond in a natural phospholipid is due to the steric hindrance brought about by the acyl, alkyl or hydroxyl group present in the sn-1 position. The lipases might also be unable to hydrolyse acyl-ester bonds involving a secondary alcohol.

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells.

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.

Studies on ether phospholipids. II. Comparative composition of various tissues from human, rat and guinea pig.

The ether phospholipid composition of various tissues (brain, heart, lung, liver, kidney, testis, erythrocytes and plasma) has been investigated in human, rat and guinea pig, using a new method of determination (El Tamer, A., Record, M., Fauvel, J., Chap, H. and Douste-Blazy, L. (1984) Biochim. Biophys. Acta 793, 213-220). This is based on the selective removal of diacyl phospholipid species by phospholipase A1 degradation followed by acidolysis of the plasmalogens. Our results fit rather well with other literature data available for human and rat tissues, illustrating the good reliability of the method. Among various differences noted between the three mammalian species, guinea pig is characterized by a relatively higher content of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and of ethanolamine plasmalogens in blood plasma. Alkylacyl-GPC, a putative precursor of platelet activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC), is also more abundant in guinea pig lung and in human kidney. This study also revealed a striking parallelism between the tissue content of alkylacyl-GPC and alkylacyl-GPE (1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine). This new observation is discussed in relation to a possible metabolic link between these two phospholipids.

Studies on enzymes related to diacylglycerol production in activated platelets. II. Subcellular distribution, enzymatic properties and positional specificity of diacylglycerol- and monoacylglycerol-lipases.

The subcellular distribution of diacylglycerol- and monoacylglycerol-lipases has been studied in human platelets. Using a fractionation procedure on Percoll gradient (Perret, B., Chap, H. and Douste-Blazy, L. (1979) Biochim. Biophys. Acta 556, 434-446), the enzyme activity displayed the same profile as that of [3H]concanavalin A, a plasma membrane marker. This result was confirmed with highly purified platelet plasma membranes prepared by adsorption onto polyethylenimine-bonded polyacrylamide beads (Kinoshita, T., Nachman, R.L. and Minick, R. (1979) J. Cell Biol. 82, 688-696). Studies with isolated membranes or crude homogenate revealed that the enzyme requires calcium or magnesium and displays an optimal pH of 6.2, showing that it is able to hydrolyse diacylglycerol under conditions where phosphatidylinositol-specific phospholipase C is fully active. Using diacylglycerol labelled in the 1- or 2-position, it was found that the two fatty acids are released at the same rate, which is supported by the lack of monoacylglycerol accumulation and by the observation that monoacylglycerol is hydrolysed at a 20-fold faster rate than diacylglycerol. Increasing concentrations of Mg-ATP promote the conversion of diacylglycerol into phosphatidic acid by diacylglycerol kinase, but only high concentrations become inhibitory for diacylglycerol lipase. These results are discussed in the light of our former hypothesis that arachidonic acid release from platelet phospholipids might occur through the sequential action of a phosphatidylinositol-specific phospholipase C coupled to a diacylglycerol lipase (Mauco, G., Chap, H., Simon, M.F. and Douste-Blazy, L. (1978) Biochimie 60, 553-561). The possible role of this enzyme in the regulation of the activity of protein kinase C is also emphasized.

Evidence for the lack of classical secretory phospholipase A2 in guinea-pig pancreas.

Several lipolytic enzymes from guinea-pig pancreas have been determined in a soluble extract and in a purified zymogen granule fractions. The positional specificity of phospholipolytic enzymes was detected using phospholipids bearing various radioactive labels. It is shown that guinea-pig pancreatic extracts are able to release both fatty acids from phosphatidylcholine, but with more efficiency towards the fatty acid occupying the 1-position of sn-glycerol. Evidence is given that guinea-pig pancreas lacks the classical secretory phospholipase A2 and that phospholipid digestion is achieved through the sequential action of phospholipase A1 and lysophospholipase.

Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas.

1. Two cationic lipases (Ia and Ib) were purified from homogenates of fresh guinea-pig pancreas by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose (twice for the latter) followed by gel filtration on Sephadex G-100. 2. Both enzymes were homogeneous upon polyacrylamide gel electrophoresis. Their molecular weights are 37,000 and 42,000 for lipases Ia and Ib, respectively, as determined by gel filtration on Sephadex G-100. Very close values for isoelectric points were found in the pH range 9.3-9.4. 3. The cationic lipases are characterized by a high phospholipase A activity (500 IU/mg protein using a potentiometric assay with egg yolk lecithin as substrate), resulting in an unusual phospholipase/lipase activity ratio of 1. 4. Using doubly labelled phosphatidylcholine, a specificity, A1, was described for the two enzymes, which are unaffected by N-ethylmaleimide, diisopropylfluorophosphate and p-bromophenacylbromide. The enzymes are insensitive to EDTA and slightly inhibited by CaCl2 and MgCl2, whereas sodium deoxycholate is required for maximal activity.