Changes in Volatile C6-Aldehydes Emitted from and Accumulated in Tea Leaves

C₆-Aldehydes emitted from intact tea leaves were analyzed quantitatively.Emission of the aldehydes increased temporarily in mid-May whenenzymatic activities involved in aldehyde formation from lipidsbegan to increase. Levels of C₆-aldehydes in tea leaves alsoincreased temporarily. However, the accumulated C₆-aldehydesdid not always correspond to emitted ones. (Received December 1, 1991; Accepted March 18, 1992)     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Seasonal Changes in Activities of Enzymes Responsible for the Formation of C6-aldehydes and C6-alcohols in Tea Leaves, and the Effects of Environmental Temperatures on the Enzyme Activities

When tea leaves were homogenized and incubated, the volatileC₆-compounds hexanal, cis-3-hexenal, cis-3-hexenol and trans-2-hexenalwere formed much more by summer leaves than by winter leavesof tea plants (Camellia sinensis). The enzymes lipolytic acylhydrolase (LAH), lipoxygenase, fatty acid hydroperoxide lyase(HPO lyase) and alcohol dehydrogenase (ADH) and an isomerizationfactor were responsible for the sequential reactions of C₆-compoundformation from linoleic and linolenic acids in tea leaf lipids,and there were seasonal changes in their activities. The tealeaf enzymes were of 3 types: LAH and lipoxygenase, which hadhigh activities in summer leaves and low activities in winterleaves; ADH, which had low activity in summer leaves and highactivity in winter ones; and HPO lyase and the isomerizationfactor, which did not seem to have any effect on the rate ofC₆-compound formation throughout the year. Changes in enzymeactivities were induced by shifts in the environmental air temperaturerather than by the age of the leaves. The combined activitiesof these enzymes determined the amounts and compositions ofthe volatile C₆-compounds formed, which are the factors thatcontrol the quality of the raw leaves processed for green tea. (Received October 6, 1983; Accepted December 20, 1983)     

Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Formation of 12-oxo-trans-10-dodecenoic acid in chloroplasts from Thea sinensis leaves

Linolenic acid-[1-¹⁴C] was converted to 12-oxo-trans-10-dodecenoic acid, via 12-oxo-cis-9-dodecenoic acid by incubation with chloroplasts of Thea sinensis leaves. Thus, it was confirmed that linolenic acid is split into a C₁₂-oxo-acid, 12-oxo-trans-10-dodecenoic acid, and a C₆-aldehyde, trans-2-hexenal, leaf aldehyde, by an enzyme system in chloroplasts of tea leaves.     

Existence of a Free Form of a Specific Membrane Lipoprotein in Gram-Negative Bacteria

The existence of a free form of a specific lipoprotein of molecular weight 7,200 was examined in the envelopes of several gram-negative bacteria. When the envelope proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, distinct peaks were observed in Salmonella typhimurium, Serratia marcescens, and Pseudomonas aeruginosa at the same position as the free form of the lipoprotein of Escherichia coli. However, the peak was not observed in Proteus mirabilis. The protein at the peak in S. typhimurium was shown to contain little or no histidine as expected from the amino acid composition of the lipoprotein. Furthermore, antiserum against the highly purified lipoprotein from E. coli was shown to react with the proteins from S. typhimurium and S. marcescens and to form the specific immunoprecipitates. In contrast, the protein from P. aeruginosa did not react with the antiserum at all. Thus, it is concluded that S. typhimurium and S. marcescens have the free form of the lipoprotein in their envelopes as does E. coli. P. aeruginosa contains a protein of the same size as the lipoprotein, but it is not certain whether the protein is the same structural protein as the lipoprotein from E. coli. P. mirabilis may not have any free form of the lipoprotein, may have it in a very small amount, or may have a lipoprotein of different molecular weight serving the same function.     

PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes

Phosphatidylinositol 4-kinase IIα (PtdIns4KIIα) localizes to the trans-Golgi network and endosomal compartments and has been implicated in the regulation of endosomal traffic, but the roles of both its enzymatic activity and the site of its action have not been elucidated. This study shows that PtdIns4KIIα is required for production of endosomal phosphatidylinositol 4-phosphate (PtdIns(4)P) on early endosomes and for the sorting of transferrin and epidermal growth factor receptor into recycling and degradative pathways. Depletion of PtdIns4KIIα with small interfering RNA significantly reduced the amount of vesicular PtdIns(4)P on early endosomes but not on Golgi membranes. Cells depleted of PtdIns4KIIα had an impaired ability to sort molecules destined for recycling from early endosomes. We further identify the Eps15 homology domain–containing protein 3 (EHD3) as a possible endosomal effector of PtdIns4KIIα. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KIIα-depleted cells. Endosomal PtdIns(4,5)P₂ was also significantly reduced in PtdIns4KIIα-depleted cells. These results show that PtdIns4KIIα regulates receptor sorting at early endosomes through a PtdIns(4)P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P₂.     

Specificity of Enzyme System Producing C6-Aldehyde in Tea Chloroplasts

The substrate specificity of enzyme system producing C₆-aldehyde in Thea chloroplasts was clarified with an entire series of synthesized positional isomers, in which the position of cis-1, cis-4-pentadiene system varies from C-3 to C-13 in C₁₈ fatty acid and geometrical isomers of linoleic acid. The structural requirement for the substrate of enzyme system producing C₆-aldehyde is the presence of cis-1, cis-4-pentadiene system between ω-6 and ω-10.     

Application of Affinity Chromatography to Lipoxygenase

A simple electronic device was constructed which, in combination with a conventional titrator, records close approximation of the buffer capacity curve (β-pH curve) for the solution of unknown composition. Since the recorded curves provide the overall picture of the distribution of weak Brönsted acids in the solution on the pK_a axis, this apparatus may be useful in various fields such as chemistry of food and agricultural products and clinical medicine, where the characterization of the complex mixtures of weak electrolytes — carboxylic acids, amono acids, proteins, amines, phenols and etc. — are important.