Lack of effect of withdrawal from cigarette smoking on theophylline pharmacokinetics.

The intravenous disposition of theophylline was determined in 12 healthy young male smokers during periods of smoking and short-term withdrawal (24 to 36 hours), using a crossover design. Median half-life, clearance, volume of distribution, hepatic extraction, and intrinsic clearance of theophylline during withdrawal were within +/- 5% of the corresponding median control (smoking) parameters and were normal in comparison with values published for smokers. The lack of change in the pharmacokinetic profile of theophylline indicates that adjustment of the dosage regimen should not be necessary immediately after smoking withdrawal.

The effect of acute withdrawal from cigarette smoking on indocyanine green and antipyrine clearance.

The effects of acute withdrawal from cigarette smoking on indocyanine green (ICG) clearance and antipyrine pharmacokinetics were studied in healthy young male volunteers. Two separate crossover clinical trials, each using 12 subjects, were used to compare the disposition of the drugs from 24 to 36 hours after withdrawal to the disposition found under control conditions. The median difference of ICG clearance and all antipyrine pharmacokinetic parameters from smoking control was less than 13%, indicating that short-term smoking withdrawal had no effect large enough to be of clinical significance on hepatic blood flow or hepatic drug-metabolizing capacity. Rates of hepatic blood flow were normal in comparison with values published for larger sample populations. The lack of any clinically significant effect of smoking withdrawal on hepatic blood flow or on the disposition of antipyrine, a drug with very low hepatic extraction, indicates that on a pharmacokinetic basis, changes in dosage regimens for most drugs are not necessary on acute withdrawal from smoking.

Effects of starvation on lung mechanics and biochemistry in young and old rats.

Two groups of rats (young and old) were food-deprived for 3 wk and were compared with age-matched fed groups. Final body weight and dry and wet weights of lungs were significantly reduced in both young and old starved rats. As determined by saline volume-pressure (VP) curves, lungs of young starved rats accepted significantly less volume at all pressure levels compared with lungs of young fed rats. When expressed as a percent of maximum lung volume, the VP curve in young starved rats was significantly shifted upward at low lung volumes. In the old rats, the VP curves were similar in fed and starved rats. Total lung content of protein, DNA, crude connective tissue, hydroxyproline, and elastin were significantly reduced in young starved compared with young fed rats, whereas in old starved rats only protein and DNA contents were lower than those in old fed animals. It appears that in rapidly growing young rats starvation leads to growth retardation, loss of connective tissue components, and possibly reduction in tissue elastic forces at low lung volumes, whereas starvation has no significant effects on lung mechanics and connective tissue in old rats.

A new procedure to analyze free fatty acids. Application to 20-mg brain tissue samples.

Fatty acids were analyzed by a new method which involved their isolation from hexane extracts of serum or brain tissue in aqueous potassium hydroxide (10 microliter) and methylation directly in this solution with methyl iodide. The resulting fatty acid methyl esters were partitioned into ethylene chloride (25 microliter) and were quantitated by gas-liquid chromatography. The procedure was documented by comparison with conventional methylation reactions on serum fatty acids. This method, which avoids thin-layer chromatography and which measures individual free fatty acid concentrations in 20-mg brain tissue samples, should be of particular value for examining regional free fatty acids in brain following ischemia and trauma.

Changes in fatty acids in phospholipids of the bronchoalveolar fluid in bacterial pneumonia and in adult respiratory distress syndrome.

Fatty acids of the phospholipid fraction of bronchoalveolar lavage fluid from patients with bacterial pneumonia or with the adult respiratory distress syndrome were chromatographed and the patterns compared with those for a control group. In the control group, palmitic acid (16:0) was the predominant fatty acid, accounting for 58.0% (SD 8.25%) of the total fatty acid, a proportion significantly higher (p less than 0.001) than in the distress-syndrome group (42.1%, SD 4.88%) or the acute pneumonia group (32.1%, SD 1.73%). There was a greater proportion of oleic acid (18:1) in the disease groups; thus the ratio of palmitic to oleic acid was useful in distinguishing these three groups. No patient with a palmitic/oleic acid ratio greater than 2.45 had evidence of parenchymal inflammation. Of those with a ratio less than 1.3, 89% had acute bacterial pneumonia.

Changes in connective tissue composition of the lung in starvation and refeeding.

Adult male rats were starved by allowing them one fifth of their measured daily food consumption until they lost 40% of their initial body weights. Some of these rats were then refed until their initial body weights were reached. We measured the total content of the following in the lung tissue of fed, starved, and refed animals: (1) elastin, (2) hydroxyproline, and (3) protein. Body weight and lung dry and wet weights were significantly reduced in starved and similar in refed rats compared with fed animals. Total contents of crude connective tissue, hydroxyproline, elastin, and protein were significantly lower in starved than in fed rat lungs. After refeeding, hydroxyproline content returned completely to levels found in fed rats, but other components only partially returned to normal values. These results provide a biochemical counterpart for our previous observations on the effects of starvation and refeeding on lung mechanics and morphologic aspects. It appears that the emphysema like changes in the lungs of starved rats are at least partly related to the loss of connective tissue elements.

Effects of starvation and refeeding on lung biochemistry in rats.

Adult rats received one fifth of their measured daily food consumption until they lost 40% body weight. Some of these rats were then refed until they reached their initial body weight. We measured the following in fed, starved, and refed animals: (1) disaturated phosphatidylcholine (DSPC) content of lung tissue and of lavage fluid, (2) protein content of lung tissue and of lavage return, and (3) DNA and RNA content of lung tissue. In starved lungs, tissue and lavage DSPC content, total protein and RNA contents, and RNA/DNA ratios were significantly lower than in fed rats. After refeeding, DSPC values returned completely to normal, whereas protein, DNA, and RNA contents were significantly higher than in fed rats. The RNA/DNA ratio was similar in the fed and refed groups. Changes in lavage DSPC are consistent with the increased surface elastic forces in starvation and their return to normal with refeeding reported by us previously. It appears that starvation leads to a reduction in cell size without changes in cell number and that refeeding is associated with a more significant increase in cell number than in cell size.

Lipogranulomas in non-fatty human livers. A mineral oil induced environmental disease.

Forty-four cases of lipogranulomas (LG) in non-fatty livers (NFL), consisting of 38 biopsies and six autopsy livers, were studied. LG in NFL have a distinct morphologic characteristic and virtually all are attached to or closely associated with the walls of hepatic venules. The reason for this peculiar location remains unexplained. Our data from lipid histochemistry and analysis of lipid extracts from the livers and foodstuffs by thinlayer chromatography and gas-liquid chromatography indicate that LG in NFL most likely represent a reaction to absorbed saturated hydrocarbons, like mineral oil, used widely in the food industry. The incidence of LG is increasing, as evidenced by a 1.7% incidence in 1952-53 compared with 4.6% in 1978-80. LG seldom present a diagnostic problem provided serial sections are examined. An awareness of the characteristic morphology will prevent an extensive granuloma work-up. They appear to be an incidental finding in liver biopsies, and of no clinical significance at present; however, their long-term implication, if any, must await future observations.

Mitochondrial cholesterol content and membrane properties in porcine myocardial ischemia.

Regional myocardial ischemia was produced in anesthetized pigs by occluding the left anterior descending coronary artery. Mitochondria were prepared from both normally perfused and ischemic myocardium after 2 h of occlusion. Mitochondria from the ischemic area exhibited an 89% increase in cholesterol content from 32.7 +/- 1.9 (control) to 62.0 +/- 0.47 (ischemic) nmol/mg protein with no change in either total phospholipid content or in membrane fatty acid composition. This increase in mitochondrial membrane cholesterol was accompanied by an increase in membrane microviscosity as indicated by increased fluorescence polarization using the fluorescent membrane probe, 1,6-diphenyl-1,3,5-hexatriene. In these same experiments the Arrhenius plot discontinuity temperature of oligomycin-sensitive adenosinetriphosphatase (ATPase) activity fell from 20.0 to 14.2 degrees C. Our results suggest that, during the myocardial ischemic process in pigs, there is an intracellular redistribution of free cholesterol that produces a marked increase in mitochondrial membrane cholesterol content. This appears to produce an altered mitochondrial membrane lipid bilayer packing, resulting in increased membrane microviscosity and, possibly, altered inner membrane ATPase function. Intracellular cholesterol redistribution may thus contribute to the cell membrane damage that occurs during the myocardial ischemic process.

Preparation of sphingolipid fatty acid methyl esters for determination by gas-liquid chromatography.

Sphingolipid fatty acids are first converted to a mixture of free acids and their n-butyl esters by heating the specimen at 85 degree C in aqueous butanolic hydrogen chloride; the butyl esters are then saponified with methanolic potassium hydroxide. After acidification and extraction into hexane, the fatty acids are extracted into a very small volume of aqueous trimethyl(m-trifluorotolyl)ammonium hydroxide (TMTFTH), injection of an aliquot of the TMTFTH extract into the gas chromatograph yields the fatty acid methyl esters by pyrolytic methylation of the quaternary ammonium salts of the fatty acids. The preparation of a specimen ready for the gas--liquid chromatographic (GLC) analysis with quantitative recovery of the sphingolipid fatty acids can be accomplished in less than 2 h. By comparison, none of a number of well-accepted techniques for the release of sphingomyelin fatty acids by hydrolysis or methanolysis released the fatty acids quantitatively in less than 3 h, and all required additional manipulations before GLC analysis.

Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis.

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.

Comparison of fatty acid composition of stable L-phase variants of Staphylococcus aureus induced by three differnet mechanisms.

The total saponifiable fatty acids of three stable L-phase variants of Staphylococcus aureus induced by cycloserine, methicillin, and lysostaphin were examined by gas-liquid chromatography. Five separate preparations of each of the three variants were examined. Twenty-nine fatty acids were identified. The fatty acid patterns of the three variants were very similary.

Simple and rapid gas-liquid chromatographic determination of diethylstilbestrol in biological specimens.

Diethylstilbestrol (DES) is readily extracted into toluene from biological fluids or tissue homogenates. Sodium carbonate is added to the initial mixture to eliminate potentially interfering substances. The toluene is extracted with a very small volume of aqueous trimethylphenylammonium hydroxide. This solution generates dimethyl DES in the vaporizer of a gas-liquid chromatograph. An internal standard, dienestrol (DI), is added at the beginning of the procedure and is partitioned and methylated in the same manner as DES. The DES and DI derivatives are well separated in less than 6 min on an ov-17 column. The entire analysis requires less than 15 min for a fluid specimen and less than 25 min for a solid tissue specimen. Seven samples can be analyzed each hour on a single column with a flame ionization detector. The relative standard deviations at levels from 2.5 to 100 ppm in bile are less than 5%. The lower limit of sensitivity is 8 ppb in a 1 ml bile sample.

Rapid determination of dipicolinic acid in the spores of Clostridium species by gas-liquid chromatography.

A gas-liquid chromatographic procedure has been developed to quantitate dipicolinic acid in bacterial spores. The culture, washed from a plate, was hydrolyzed with acid containing the internal standard, pyridine-2,4-dicarboxylate, and then extracted into methyl isobutyl ketone. The internal standard and dipicolinic acid were then extracted into a small volume of trimethylphenylammonium hydroxide. Injection of the resultant quaternary ammonium salts into a gas chromatograph yielded, via thermal decomposition, the methyl ester derivatives of the dipicolinic acid and the internal standard. The amount of dipicolinic acid in the sample was determined from a standard curve. The method was sensitive to 100 ng of dipicolinic acid per sample and was 1,000 to 5,000 times more sensitive than the commonly used methods. Preparation of the sample required less than 1.5 h and less than 15 min of the analyst's time.

A simple, rapid method for measurement of acetate in tissue and serum.

A simple and rapid method is described for determining the free acetate concentration in liver and serum. After extraction, the acetate is converted to its benzyl ester by thermal degradation of its benzyldimethylphenylammonium salt in the vaporizer of a gas chromatography. Good quantitation is achieved in the range of 0.033-2.5 mumoles of acetate per gram of liver or per milliliter of serum.