Human pancreatic chymotrypsinogen A: a non-competitive enzyme immunoassay, and molecular forms in serum and amniotic fluid.

A sandwich enzyme immunoassay has been developed for human pancreatic chymotrypsin(ogen) using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labelled IgG as the second antibody. The detection limit was 0.5 microgram/l. A good parallelism was observed with the curves obtained from standard chymotrypsinogen A and chymotrypsin(ogen) present in pancreatic juice; however, a slight discrepancy in parallelism with chymotrypsin(ogen) present in serum and amniotic fluid was noticed. Chymotrypsinogen concentration in pancreatic juice was evaluated to represent 9% of total proteins. Mean values of chymotrypsin(ogen) in human sera were 24.6 +/- 8.3 micrograms/l in adults and 20.9 +/- 8.8 micrograms/l in newborns. In amniotic fluid at the 18th week of pregnancy the values were scattered (5-70 micrograms/l). The molecular forms of immunoreactive chymotrypsin(ogen) in normal serum and amniotic fluid have been investigated by gel filtration on Sephadex G-100. Two peaks of immunoreactive chymotrypsin(ogen) were observed in normal serum; the first peak elutes in a position consistent with a complex of chymotrypsin with serum inhibitor (Mr 76,000), and the second peak elutes with a molecular weight of approx. 25,000 corresponding to the elution position of free chymotrypsin(ogen). In normal amniotic fluid three peaks of immunoreactive material were present; the first and second peaks elute in the same position as in serum, and the third peak with a molecular weight of about 14,500 may represent a degraded form of chymotrypsin.

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.

Individual and combined effects of low oral doses of deoxynivalenol and nivalenol in mice.

Deoxynivalenol (DON) and nivalenol (NIV) are toxic Fusarium secondary trichothecene metabolites that often co-occur regularly in cereal grains. These compounds were compared for their toxicity towards C57BL/6 mice on several parameters including alteration in plasma biochemistry, immune system reactivity and hepatic drug metabolism capacity. Mice received individual or combined oral doses of each toxin: 0.071 or 0.355 mg/kg of body weight, administrated three days a week for 4 weeks. Food consumption was altered by the single administration of 0.355 mg/kg of NIV, although no noticeable change of body and organ weights or liver protein contents was detected. NIV administration did cause also significant changes in total CO2 and uric acid concentrations in plasma. Individual toxin exposures led to increases in plasma IgA without no detectable change in the ex vivo production of cytokine by splenocytes. The liver ethoxyresorufin O-deealkylase, pentoxyresorufin O-depenthylase and glutathione S-transferase activities were increased in concert with cytochrome P4501a and P4502b subfamily expression. Administration of combinations of DON and NIV resulted in responses similar to that observed using individual doses of each toxin. However, depending on the ratio of toxin doses and biochemical parameters, some responses could be also additive (plasma IgA and hepatic DCNB conjugation) or synergistic (plasma uric acid).

Biological validation of coenzyme Q redox state by HPLC-EC measurement: relationship between coenzyme Q redox state and coenzyme Q content in rat tissues.

The properties of coenzymes Q (CoQ9 and CoQ10) are closely linked to their redox state (CoQox/total CoQ) x 100. In this work, CoQ redox state was biologically validated by high performance liquid chromatography-electrochemical measurement after modulation of mitochondrial electron flow of cultured cells by molecules increasing (rotenone, carbonyl cyanide chlorophenylhydrazone) or decreasing (antimycin) CoQ oxidation. The tissue specificity of CoQ redox state and content were investigated in control and hypoxic rats. In control rats, there was a strong negative linear regression between tissular CoQ redox state and CoQ content. Hypoxia increased CoQ9 redox state and decreased CoQ9 content in a negative linear relationship in the different tissues, except the heart and lung. This result demonstrates that, under conditions of mitochondrial impairment, CoQ redox control is tissue-specific.

[Phospholipase of rat intestine: mode of action]. / Phospholipases de l'intestin de rat: mode d'action.

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.

Non-competitive enzyme immunoassay of human trypsin 1.

A sandwich enzyme immunoassay was developed for human pancreatic trypsin 1 using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labeled IgG as the second antibody. The entire assay takes 6 h and the detection limit is 0.5 microgram/l. The assay can be performed on sera samples or on discs carrying dried blood spots. Good agreement was found with a radioimmunoassay kit. This simple assay could be widely applied to confirm the elevated immunoreactive serum trypsin described in newborn children with cystic fibrosis.

Tolerance and biological activity of pentosan polysulfate after intramuscular or subcutaneous administration for ten days in human volunteers.

This study reports on the tolerance and the pharmacological activity of pentosan polysulfate (PPS) administered to healthy volunteers for 10 days. Three groups of 10 subjects received either one daily injections of 100 mg of PPS by I. M. route (group I), or two daily injection of 50 mg of PPS by I. M. or S. C. route (groups II and III, respectively). In each group two random subjects received a placebo for the 10 days; on day 0, each subject was injected by a placebo. Clinical tolerance was checked by a daily physical examination; biological tolerance was assessed comparing the results of the main biochemical and haematological constants measured before starting the treatment (day 0) and 12 or 24 h after the end of the treatment (day 11). The pharmacological activity was measured on serial samples taken before treatment and between 1 and 6 h after the drug injection on days 1, 3 and 10; the results were compared to those obtained on day 0. Clinical tolerance was good. The biological side effects concern the transaminase levels and the platelet counts. An increase above the upper normal limit was observed in 18/24 and 3/24 for alanine and aspartic transaminase respectively. The mean platelet reduction ranged between 24 and 34% according to the groups. The drug injection induced a slight Quick time (PT) prolongation, no significant alteration of factors II, VII-X, V levels and of thrombin clotting time. The activated partial thromboplastin time (APTT) was significantly prolonged and there was a weak but significant circulating anti-Xa activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal changes related to eating behavior in oligomenorrheic women.

The aim of this study was to determine those hormonal alterations in the gonadotropin-ovarian axis that are related to eating behavior in oligomenorrheic patients. We studied 74 oligomenorrheic women aged 26.2 +/- 0.8 years, divided into group IA (N = 13) with eating disorders, group IB (N = 61) without eating disorders and 18 normally cycling controls aged 29.2 +/- 1.6 years (group II). No subject had ovarian failure, pituitary disease, thyroid dysfunction or was taking any drug. Blood samples were taken on days 3-6 after the last menses. Luteinizing hormone (LH) was measured in two plasma pools, each made up of three samples taken at 30-min intervals, starting at 15.00 h (LH-15h) and 09.00 h (LH-9h), which allowed the mean LH (mLH) and variability in LH (V-LH: percentage increase from the lower to the higher of the two LH values) to be calculated. Follicle-stimulating hormone (FSH), sex steroids, and gonadotropin-releasing hormone-stimulated LH (sLH) and -FSH (sFSH) were also evaluated. Eating behavior was evaluated with the EAT questionnaire; the EAT 26 score, the dieting score (DS) and bulimia score (BS) were calculated. Dietary intake was evaluated in 35 group IB patients based on food diaries analyzed with the REGAL program, to evaluate daily calorie intake (Cal) and calories provided by carbohydrates (Carb), lipids (Lip) and proteins (Prot). Comparisons between groups were done by analysis of variance (followed by the Fisher PLSD test) and the Kruskal-Wallis test. Groups IA, IB and II did not differ regarding age, body mass index, LH-9h, LH-15h, mLH, FSH, sLH, sFSH, estradiol or dehydroepiandrosterone sulfate; group IA had a higher V-LH than group II (p < 0.02) and a higher testosterone level than groups IB and II (p < 0.05). Positive correlations were found between V-LH and DS (p < 0.01) and BS (p < 0.05), and between testosterone and BS (p < 0.02) and DS (p < 0.05). The V-LH was negatively correlated with Cal and Carb, and testosterone was positively correlated with Cal and Lip. In patients referred for oligomenorrhea, it is concluded that testosterone levels and variability of LH levels are related to eating behavior.

The diagnostic value of acetylcholinesterase/butyrylcholinesterase ratio in Hirschsprung's disease.

Aganglionosis of large bowel (Hirschsprung's disease; HD) is associated with higher acetylcholinesterase activity (AChE activity). Occasionally, especially in the neonatal period, the AChE activity may not be of diagnostic value. The authors previously reported that simultaneous estimation of butyrylcholinesterase activity (BChE activity) and the determination of AChE/BChE ratio may have discriminatory diagnostic value. They extended this finding to 31 cases of HD, in 16 of which resected tissue was available for study. All cases had histologic confirmation of aganglionosis. The AChE/BChE ratio was found to be higher than 2.0, with the exception of a case in which the biopsy weight was low (i.e., less than 3 mg), even when the AChE activity was normal or borderline. The estimation of AChE/BChE ratio is easy, rapid, and, in the author's experience, of discriminatory diagnostic value.

Copper-ligand interactions and physiological free radical processes. Part 2. Influence of Cu2+ ions on Cu(+)-driven .OH generation and comparison with their effects on Fe(2+)-driven .OH production.

In our search to establish a reference .OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu2+ ions on the Cu+/H2O2 reaction has been investigated. This influence depends on the CCu2+/CCu+ ratio. At low Cu2+ concentrations, .OH damage to various detector molecules decreases with increasing Cu2+ concentrations until CCu2+/CCu+ reaches unity. Above this value, .OH damage increases sharply until CCu2+/CCu+ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu2+ cancels. Finally, at higher concentrations, Cu2+ ions logically add their own .OH production to that normally expected from Cu+ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu+ ions on the generation of .OH radicals by water gamma radiolysis has also been tested and, as already established for Cu2+ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge .OH radicals in the absence of a ligand.

Cholinesterase activities in resected bowel specimens from children with Hirschsprung's disease.

We have measured acetylcholinesterase (AChE) and the butyrylcholinesterase (BChE) activities along strips of resected bowel specimens from thirteen children with Hirschsprung's disease. The AChE activity was variable along the length of the resected bowel, while the BChE activity remained constant. By contrast, the AChE/BChE ratio decreased rapidly to low levels when ganglion cells were present. We suggest that this ratio can be used to determine the normal segments of the bowel, to specify the anatomic forms (i.e. rectal or rectosigmoid) and may be useful in determining the anastomotic level.

Assay of human pancreatic lipase in biological fluids using a non-competitive enzyme immunoassay.

A sandwich enzyme immunoassay has been developed for human pancreatic lipase using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labelled IgG as the second antibody. The detection limit was 0.5 microgram/l. Good parallelism was observed with the curves obtained from standard lipase and lipase present in serum, pancreatic juice and duodenal contents, demonstrating that the assay may be used to measure the level of the protein in different biological fluids. Mean values of lipase in human sera were 12.3 +/- 6.8 micrograms/l in adults and 4.5 +/- 2.7 in newborns. In all cases a good correlation was found in serum between the catalytic activity and the enzyme immunoassay. Lipase is detectable in amniotic fluids at the 18th week of pregnancy but at a very low level (0.95 +/- 0.32 microgram/l). In pancreatic juices, lipase concentration was 14.6% of the total protein content. A study on cystic fibrosis patients showed a poor correlation between blood pancreatic lipase concentration and fat malabsorption underlying the difficulty in assessing pancreatic function by the measurement of serum pancreatic enzymes. The use of the lipase assay in duodenal contents would permit better assessment of pancreatic function in patients presenting a severe or borderline defect in fat digestion and absorption.

Fatty acid composition and kinetic behaviour of liver retinyl esters in vitamin A sufficient and deficient rats.

Weanling rats were fed vitamin A deficient diets (-A) or diets supplemented with vitamin A (+A) (4.4 mg retinol equivalents/kg diet) for a period of 7 or 6 wk, respectively. In liver tissues of these two groups of animals both the subcellular localization as well as the fatty acid composition of the retinyl esters was studied. During vitamin A supplementation or deprivation, the kinetics of the different ester forms were investigated. Results indicate that the subcellular localization of all retinyl esters is similar and dependent on age. Two pools exist, ie one consisting of the nuclear/cell debris and mitochondrial-lysosomal fractions and the other containing the microsomal and cytosol fractions. HPLC analysis showed retinyl palmitate as the predominating (80%) form of the various retinyl esters. By supplementation clearly two kinetic behaviours can be demonstrated: one being a relatively stable storage of the palmitate and stearate, increasing with time and the second one being a more labile pattern for the ester forms with other saturated and unsaturated fatty acids. By vitamin A depletion all retinyl esters are affected indicating that the ester forms other than palmitate and stearate are also storage forms of vitamin A.

Increased concentrations of endogenous 13-cis- and all-trans-retinoic acids in diffuse idiopathic skeletal hyperostosis, as demonstrated by HPLC.

Endogenous 13-cis- and all-trans-retinoic acids have been quantitated in human serum using a solvent extraction procedure followed by isocratic reversed phase high performance liquid chromatography and UV detection. In healthy adults, after an overnight fasting period, the concentrations of 13-cis- and all-trans-retinoic acids yielded 5.3 +/- 2.43 nmol/l and 11.8 +/- 3.3 nmol/l, respectively (mean +/- SD). The method has been successfully applied to the analysis of both isomers in serum from patients with idiopathic skeletal hyperostosis in whom, the 13-cis- as well as all-trans-retinoic acid levels were raised as compared to the control group.

In vivo effects of olive oil-based lipid emulsion on lymphocyte activation in rats.

Numerous studies suggest that immune function may be compromised by lipid emulsions rich in polyunsaturated fatty acids, especially linoleic acid. In our study, we compared the effect of a new olive oil-based lipid emulsion (ClinOleic(R)) containing 18% linoleic acid, and an emulsion based on soybean oil (Ivelip(R); 52% linoleic acid) on lymphocyte functions. Weaning Wistar rats (n= 24) were fed for 4 weeks on an oral diet that contained 12% of total energy as lipids from soybean oil. Then they received, during 6 days, a total parenteral nutrition (260 kcal/kg/d) in which 12% of total energy was brought by one of the two lipid emulsions. The fatty acid profile of spleen lymphocyte phospholipids reflected lipid intakes, with a higher content of oleic acid in ClinOleic(R) group and linoleic acid in Ivelip(R) group. A greater proportion of cells expressed the interleukin-2 receptor a-chain (CD25) after administration of ClinOleic(R) when compared to Ivelip(R) (55.43 +/- 3.47 vs 45.48 +/- 3.26%, P<< 0.05). Moreover, the CD25 expression was positively correlated with oleic acid content of spleen lymphocyte phospholipids (r= 0.500, P<< 0.018). These results show that ClinOleic(R) is able to induce, in vivo, a greater proportion of cells expressing CD25, and suggest that oleic acid could have a role in the observed effects.

Metabolic utilization of linoleic acid from fat emulsion in infants during total parenteral nutrition.

The metabolic utilization of linoleic acid (C18:2w6) from intravenous (iv) soybean oil emulsion via its conversion to higher homologues and prostaglandin biosynthesis was studied in 21 infants. The infants were of normal birth weight, in good clinical and metabolic condition, and aged from 1-4 months. They all received total parenteral nutrition after birth for more than 1 month; the only lipid was provided by iv Intralipid 20%, at levels approximating 350 mg/kg/24 h of linoleic acid (2.5% of the infants' total daily energy intake). Study of the fatty acids of plasma and erythrocyte phospholipids in these infants, compared to healthy controls of the same age (N = 26), showed that 350 mg/kg/24 hr of C18:2w6 supply resulted in normalization of erythrocyte phospholipid fatty acid distribution. This was particularly true of the higher homologues of C18:2w6 (C18:3w6, C20:3w6, and C20:4w6). This suggested that delta 6 and delta 5 desaturation activities are normal in these conditions with this C18:2w6 supply. These results were confirmed by the study of 24-hr urinary excretion of prostaglandins PGE1 and PGE2. With 350 mg/kg/24 hr of C18:2w6 supply their urinary levels were not significantly different from those observed in normal children of the same age receiving an equivalent oral C18:2w6 intake.

Plasma phospholipid fatty acids and urinary excretion of prostaglandins PGE1 and PGE2 in infants during total parenteral nutrition, with continuous or sequential administration of fat emulsion.

During total parenteral nutrition, using an identical supply of fat emulsion (350 mg/kg/24 hr) to correct essential fatty acid deficiency in children, the efficacy of two methods of administration was studied: continuous over 24 hr, or discontinuous 3 hr/day. At the beginning of the study, all the infants (1-4 months old) had proven essential fatty acid deficiency. After at least 1 month of one of the two nutritional protocols (continuous or discontinuous), plasma phospholipid fatty acid composition and PGE1 and PGE2 urinary excretion were measured. The results obtained indicate better utilization of the fat emulsion when it is administered almost every day, in continuous infusion over 24 hr (1 g/kg/24 hr of Intralipid 20%).

Fatty acid composition of adipocyte membrane phospholipids and stored triglycerides in infants receiving total parenteral nutrition.

Fatty acid (FA) composition of membrane phospholipids (PL) and stored triglycerides (TG) from adipose tissue was studied in eight infants aged 1 to 4 months receiving total parenteral nutrition (TPN) since birth. During this period, essential fatty acid (EFA) intake consisted exclusively of soybean oil emulsion administered by intravenous route (Intralipid 20%) representing 301 +/- 88 mg/kg/24 hr of linoleic acid and 58 +/- 18 mg/kg/24 hr of alpha-linolenic acid, or 2.3 +/- 0.6% and 0.4 +/- 0.1%, respectively, of total energy intake. The results were compared with those of eight control infants of the same age receiving orally a normal milk diet with an intake of 660 +/- 260 mg/kg/24 hr of linoleic acid and 101 +/- 35 mg/kg/24 hr of alpha-linolenic acid, or 4.5 +/- 0.7% and 0.7 +/- 0.3%, respectively, of total energy intake. Although their EFA intake was significantly lower (p less than 0.01) and administered only parenterally, after 1 to 4 months the infants receiving TPN still had a membrane phospholipid FA pattern of adipose tissue which was not significantly different from that of normal children of the same age. In stored adipocyte TG, the percentage of linoleic acid was significantly lower (p less than 0.01) in infants receiving TPN. This is probably of nutritional importance as at this stage of life the child builds up its stores of EFA. The proportion of the other fatty acids in adipocyte TG was not significantly modified.

Total parenteral nutrition decreases liver oxidative metabolism and antioxidant defenses in healthy rats: comparative effect of dietary olive and soybean oil.

BACKGROUND: Total parenteral nutrition (TPN) is used for critically ill patients undergoing surgery, after trauma, or during disease conditions that favor oxidative stress. We studied the effect of TPN on liver oxidative metabolism and antioxidant defenses in rats, and we compared the effect of soybean oil- and olive oil-based diets. METHODS: Seven-week-old rats (n = 28) were divided into four groups. Two experimental groups received a TPN solution containing soybean oil (TPN-S) or a mixture of olive/soybean oil, 80/20 (TPN-O), IV for 6 days. Orally fed animals received a solid diet including soybean oil (Oral-S) or olive/soybean oil, 80/20 (Oral-O). The following parameters were measured: DL-alpha-tocopherol, vitamin A, malondialdehyde and thiobarbituric acid reactive substances (MDA-TBARS), and total radical-trapping antioxidant parameter (TRAP) in serum; DL-alpha-tocopherol, vitamin A, glutathione (GSH), and catalase (Cat) activity in liver homogenate; fatty acids from phospholipid, cytochrome P-450 content, NADPH-cytochrome c2 reductase activity in liver microsomes; superoxide dismutase (SOD), glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione transferase (GST), and glucose-6-phosphate dehydrogenase (G6PD) in liver cytosol. RESULTS: The soybean or olive oil diets modified the liver microsomal fatty acid phospholipid composition, but the unsaturation index remained unchanged. TPN specifically increased the saturation of the membrane. The cytochrome P-450 level and the NADPH-cytochrome c2 reductase, SOD, Gpx, Cat, and GST activities were unchanged by soybean oil or olive oil diet but decreased receiving TPN. CONCLUSIONS: In rats, TPN decreased the liver oxidative metabolism and enzymatic antioxidant defenses. This may be related to saturation of the liver microsomal fatty acids.