Recovery from laparoscopic cholecystectomy procedures.

The authors studied the postoperative experiences of 53 patients who had uncomplicated laparoscopic cholecystectomy procedures. Patients rated their pain, nausea, vomiting, and fatigue before surgery, before discharge, and on postoperative days one, two, three, four, and seven. The majority of patients reported more difficult and painful and slower recoveries than they expected or that they believed were indicated in the education materials provided to prepare them for surgery. The experiences of the patients in this study clearly indicate a need to modify preoperative preparatory education materials.

Spectrophotometric assays for the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/arylesterase.

Human serum plasma paraoxonase/arylesterase exhibits a genetic polymorphism for the hydrolysis of paraoxon. One allelic form of the enzyme hydrolyzes paraoxon slowly with a low turnover number and the other(s) hydrolyzes paraoxon rapidly with a high turnover number. Chlorpyrifos-oxon, the active metabolite of the insecticide chlorpyrifos (Dursban), is also hydrolyzed by plasma arylesterase/paraoxonase. A specific assay for measuring hydrolysis of this compound is described. This assay is not subject to interference by the esterase activity of serum albumin. The Km for chlorpyrifos-oxon hydrolysis was 75 microM. Hydrolysis was inhibited by phenyl acetate, EDTA, and organic solvents. Enzyme activity required calcium ions and was stimulated by sodium chloride. Hydrolysis was optimized by using methanol instead of acetone to dissolve substrate. Unlike the multimodal distribution of paraoxonase, the distribution of chlorpyrifos-oxonase activity failed to show clear multimodality. An improvement in the assay for hydrolysis of paraoxon by plasma arylesterase/paraoxonase was achieved by elimination of organic solvents. Plotting chlorpyrifos-oxonase activity vs paraoxonase activity for a human population using the new assay conditions provided an excellent resolution of low activity homozygotes from heterozygotes for this allele. A greater than 40-fold difference in rates of chlorpyrifosoxon hydrolysis observed between rat (low activity) and rabbit sera (high activity) correlated well with the reported large differences in LD50 values for chlorpyrifos in these two animals, consistent with an important role of serum paraoxonase in detoxification of organophosphorus pesticides in vivo.

Primary structure of four allatostatins: neuropeptide inhibitors of juvenile hormone synthesis.

Four neuropeptides that inhibit juvenile hormone synthesis by the corpora allata have been isolated from brains of the virgin female cockroach Diploptera punctata. These allatostatins are 8-13 amino acids long, are amidated, and show sequence similarity, including a 3-amino acid sequence at the C-terminal end that is common to all four peptides. The peptide sequences are as follows: allatostatin 1, Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2; allatostatin 2, Gly-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu-NH2; allatostatin 3, Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2; and allatostatin 4, Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2. An in vitro bioassay of the synthesized allatostatins showed greater than 40% inhibition of juvenile hormone synthesis by corpora allata of virgin females with 10(-9) M allatostatin 1, 10(-8) M allatostatins 2 and 4, and 7 X 10(-7) M allatostatin 3. Inhibition by allatostatins 1-4 was reversible. In addition, allatostatin 1 inhibited juvenile hormone synthesis by corpora allata from mated females and last-instar larvae of D. punctata and corpora allata of adult female Periplaneta americana.

Role of genetic polymorphism of human plasma paraoxonase/arylesterase in hydrolysis of the insecticide metabolites chlorpyrifos oxon and paraoxon.

Plasma paraoxonase is a polymorphic enzyme that hydrolyzes paraoxon, the neurotoxic, active metabolite of the insecticide parathion. This enzyme is specified by at least two alleles with frequencies of about .7 and .3 among Caucasoid populations. A specific assay was developed that measured the activity of human plasma paraoxonase without interference from serum albumin which contributes significantly to the hydrolytic breakdown of paraoxon at the high pH values used in many previous assays. There was an 11-fold variation in paraoxonase activities, and the population distribution was at least bimodal. However, this specific assay did not improve the discrimination between the three genetic classes: (1) homozygotes for the low-activity allele, (2) heterozygotes, and (3) homozygotes for the high-activity allele. Chlorpyrifos oxon--the neurotoxic metabolite of the organophosphorus insecticide chlorpyrifos (Dursban)--was hydrolyzed by the same plasma fraction that hydrolyzed paraoxon. There was only four- to fivefold variability in enzyme activity, and the population distribution was unimodal. Homozygotes for low paraoxonase activity ranged over almost the entire spectrum of chlorpyrifos oxonase activity. Possible differences in susceptibility to chlorpyrifos toxicity therefore are unlikely to be predicted by the paraoxonase genotype alone. The ratio of paraoxonase over that of chlorpyrifos oxonase provided an excellent method for genetic typing of the paraoxonase polymorphism, as did the substitution of phenylacetate for chlorpyrifos as the substrate.

Immunoquantitation of cytochrome b5 and methylcholanthrene-induced cytochromes P-450.

The enzyme-linked immunosorbent assay (ELISA) has been investigated for its ability to quantitate hydrophobic proteins like cytochromes b5 and P-450 at the subnanogram level. Issues encountered that have broad significance not only for ELISA, but for other qualitative and quantitative immunoassays as well, include the effects of detergent, the discriminatory capacity of ELISA, and the method for determining an assay's selectivity.

Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats.

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay.

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.

An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein.

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.