Determination of sulfated and nonsulfated bile acids in serum by mass fragmentography

A Sep-Pak C18 cartridge was used for purification of bile acids from serum. Three kinds of deuterium labeled internal standards were required for accurate measurement of individual sulfated and nonsulfated bile acids. These internal standards were added to the serum before its application to the cartridge. Separation of sulfated and nonsulfated bile acids was performed on piperidinohydroxypropyl Sephadex LH-20 column chromatography. The nonsulfate fraction was submitted to alkaline hydrolysis, and the sulfate fraction to solvolysis followed by alkaline hydrolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivatives and quantitated by mass fragmentography. The recovery of each bile acid sulfate was quite satisfactory. In fasting healthy subjects the mean of total nonsulfated bile acids in serum was 1.324 micrograms/ml, and that of total sulfated bile acids was 0.450 micrograms/ml. Sulfated lithocholic acid comprised a large part of sulfated bile acids in healthy subjects.     

Characterization of partially purified prostaglandin E2 receptor from the canine renal medulla: evidence for physical association of the receptor protein with the inhibitory guanine nucleotide-binding protein.

Prostaglandin (PG) E2 binding protein, a putative PGE2 receptor, was purified 26-fold with 0.4% recovery from canine renal outer medullary membranes solubilized with 12% digitonin with the sequential use of a Superose 12, Wheat Germ Agglutinin (WGA) Affigel 10, DEAE-5PW and Ampholine column chromatographies. The final preparation retained the binding activity specific for PGE2, but lost most of the sensitivity to guanosine-5'-(gamma-thio)triphosphate (GTP gamma S). An antibody against alpha subunit of the inhibitory guanine nucleotide-binding protein (alpha Gi)1 and alpha Gi2 or that against common sequences of alpha subunit of guanine nucleotide-binding proteins (alpha G(common)) reacted at 41 kDa protein in the sample of each step of purification, but failed to do so in the final preparation. An antibody against alpha Gi3 or alpha Go had no effect. In fact, peaks of the binding activity and immunoreactivity for alpha Gi1,2 were chromatographically separated by isoelectric focusing. Moreover, antibodies against alpha G(common) or alpha Gi1,2, but not that against alpha Gi3 and alpha Go, precipitated PGE2 binding activity in the active fractions of WGA-Affigel 10 column chromatography. These results suggest that the PGE2 receptor is an acidic glycoprotein and that Gi1 or Gi2 is physically associated with the PGE2 receptor and dissociates from the receptor protein during purification procedures.     

Two pathways for GM2(NeuGc) expression in mice: genetic analysis

We have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GalNAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concerning the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42: 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(NeuGc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

Stereospecificity of hydrogen transfer by pyridine nucleotide-dependent enoyl reductases in fatty acid synthesis: Studies with enzymes obtained from developing castor bean seeds and Chlorella vulgaris

The mechanism of hydrogen incorporation into fatty acids wasinvestigated with fatty acid synthetase systems from developingcastor bean seeds and Chlorella vulgaris. Fatty acids synthesizedin the presence of D₂O or stereospecifically deuterium-labeledNADPH or NADH were isolated and analyzed by mass spectrometryto examine the localization of deuterium atoms in the molecule.The stereospecificities of ß-ketoacyl-acyl carrierprotein (ACP) reductase and enoyl-ACP reductase for reducedpyridine nucleotide were determined with acetoacetyl-ACP andcrotonyl-ACP as substrates. The products were also analyzedby gas chromatography-mass spectrometry. The following resultswere obtained:

1. ß-Ketoacyl-ACP reductases from both castor bean seedsand C. vulgaris used the B-side hydrogen of NADPH.
2. Enoyl-ACPreductase from C. vulgaris required NADH for the activity.
3. Enoyl-ACPreductase from castor bean seeds used the A-side hydrogenofNADPH, whereas that from C. vulgaris used the B-side hydrogenof NADH.
4. When stearate was synthesized with the crude fattyacid synthetasefraction from castor bean seeds, hydrogen atomsfrom water werefound on the even-numbered methylene carbonatoms (two hydrogenatoms per carbon atom) and some were foundon the odd-numberedmethylene carbon atoms. Hydrogen atoms fromthe B-side of NADPHwere found on the odd-numbered methylenecarbon atoms (one hydrogenatom per carbon atom). Hydrogen atomsfrom the A-side of NADPHwere also found on the odd-numberedmethylene carbon atoms,but the number of incorporated hydrogenatoms was less thanexpected.

(Received October 17, 1979; )     

Identification of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in chronic subdural hematoma.

We detected a novel kind of bile acid in the content of chronic subdural hematoma. This substance was specifically found in chronic subdural hematoma, and not in subdural hygroma, which is pathologically similar except for the lack of capsular membrane. The compound was identified as 7 alpha-hydroxy-3-oxo-4-cholestenoic acid by high performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectrometry. The structure was confirmed by the comparison with the chemically synthesized compound. The average contents in chronic subdural hematoma were 658.09 +/- 137.53 ng/ml, while those in normal human plasma were 126.27 +/- 17.73 ng/ml. It was not detected in normal cerebrospinal fluid. The higher level in chronic subdural hematoma than human plasma strongly suggests the local, extrahepatic production of this type of C27 bile acids.     

Immunological quantitation and immunohistochemical localization of leukotriene A4 hydrolase in guinea pig tissues

We prepared a highly specific polyclonal antibody against leukotriene (LT) A4 hydrolase using a recombinant human enzyme. Using this antibody, we quantified LTA4 hydrolase protein content in the cytosols of guinea pig tissues. The enzyme protein content correlated well with the enzyme activity with a correlation coefficient of 0.87. However, the enzyme activity per mg of the enzyme in the cytosols was low, particularly in the liver and adrenal gland, compared with the specific activity of the purified enzyme. These observations suggest the presence of inhibitory substances and/or inactive enzymes in the cytosols of these tissues. To determine the cellular localization of LTA4 hydrolase in tissues other than blood cells, we carried out immunohistochemical examinations of guinea pig tissues. We identified epithelial cells in the tracheobronchial system and gastrointestinal tract, smooth muscle cells in the bronchi and aorta, vascular endothelial cells, and the intestinal plexus as novel cellular sources of the enzyme in the parenchyme of the tissue. Thus, LTA4 hydrolase was widely distributed in various types of parenchymal cells in the tissues, and this observation warrants further investigations on the biological activities of LTB4 in these cells and tissues.     

Identification of novel decenoic acids in heated butter

Novel decenoic acids such as (E)-4-decenoic acid and (E)- and (Z)-5-,6-decenoic acid were detected as minor components in heated butter using GC and GC/MS. The formation mechanism of these novel decenoic acids is discussed on the basis of the result of the reaction of delta-decalactone with active clay in a model experiment.