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To assess the postmortem stability of brain 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) and 3,4-dihydroxyphenylethyleneglycol (DHPG) levels, groups of rats and mice were killed by cervical dislocation and left at either 21 degrees or 4 degrees C for intervals of up to 24 h until removal and freezing of whole brain. Whole brain free and total MHPG and DHPG levels were determined simultaneously by gas chromatography-mass fragmentography (GC-MF). By 2 h death, statistically significant decrements occurred in rat brain free DHPG (20%), total MHPG (21%), and total DHPG (11%) at 4 degrees C, but free MHPG increased significantly (50%) compared with controls. At 21 degrees C, rat brain total MHPG increased compared with controls at 2 h (15%) but decreased at 4 h (15%) and 8 h (15%), whereas free MHPG levels were increased at these times. Although brain total and conjugated DHPG levels showed little change, free DHPG levels were reduced at all times. In mouse brain no significant changes occurred in free MHPG and DHPG by 24 h at 4 degrees C. At 21 degrees C, mouse brain DHPG levels decreased whereas MHPG concentrations increased over the 8-h period of study. These findings demonstrate the occurrence of significant postmortem time- and temperature-dependent changes in brain MHPG and DHPG concentrations and indicate caution in the interpretation of changes in these metabolites in studies employing human postmortem brain tissue.

Use of prometaphase chromosome preparations has led to significant improvements in the localization of both NORs and ribosomal gene clusters in the short arms of human acrocentric chromosomes. An improvement of the NOR silver-staining method, followed by trypsin-Giemsa banding, was used to identify the precise location of the NOR on each human acrocentric chromosome. For comparison, the satellite, stalk, and centromeric region were also identified with the aid of both Q- and G-banding techniques. The amount of silver impregnation present in the stalk region of the D- and G-group chromosomes was unique for each of these acrocentric chromosomes and depended on the length of the stalk. In situ hybridization was used to locate the ribosomal gene clusters. A plasmid containing 5.6 kb of the 18S rDNA gene was first oligolabeled with bio-16-dUTP, then hybridized in situ to metaphase chromosomes and visualized by an alkaline phosphatase color-detection system. Our results indicated that, in most cases, the location of the 18S rDNA gene cluster in the stalk region was indistinguishable from the site of silver impregnation. However, exceptions were noted, suggesting a multiplicity of arrangements of the ribosomal gene clusters. A model is proposed to describe the spatial relationship of NORs (transcriptional activity) and the ribosomal gene clusters.

While chorionic villus sampling allows both early and rapid prenatal diagnosis of chromosome disorders, the accuracy of this technique has not been fully established. Maternal cell contamination and pseudomosaicism represent two major sources of diagnostic error. Combined use of both direct chromosome preparations and villus cultures is important in overcoming these problems. Direct preparations of villus tissue allow recognition of maternal cell contamination of villus cultures. Conversely, villus cultures yield higher resolution chromosomes and may be helpful in differentiating between true versus pseudomosaicism when two or more cell lines are identified in direct chromosome preparations. Preliminary data suggest that analysis of direct preparations from multiple individually processed villus fragments may also be of value in this regard. Until more experience is gained, mid-trimester amniocentesis should be offered to CVS patients when mosaicism is encountered.

A gas chromatographic-mass fragmentographic (GC-MF) procedure is described for the simultaneous quantitation of 3,4-dihydroxyphenylethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl-trifluoroacyl esters, using [2H2]DHPG and [2H3]MHPG as internal standards. Assay sensitives of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 +/- 4.11 ng/g) and free (13.0 +/- 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 +/- 3.70 ng/g) and free (12.3 +/- 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (greater than 90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and efflux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.

The objectives of this research were to determine optimal conditions for culturing trophoblast cells in vitro and to ascertain whether cells maintained under these conditions are an adequate source of material for fetal chromosome studies. Best results were obtained with the use of 1.25 X 10(6) cells in a total volume of 4 ml of M-199 medium that contained 20% calf serum and 1 mg/ml glucose, incubated in an atmosphere of 95% air and 5% carbon dioxide. Cytogenetic analysis was performed in trophoblast cultures 14 to 64 days old. In all cases it was possible to identify cells in metaphase which were adequate for karotyping. Evidence that the cultured cells were fetal in origin was provided by two cases in which chromosome abnormalities--46,XY,inv(18), and 47,XX, + 21--were identified both in the placental cultures and in leukocytes and fibroblasts from the fetus. The cytogenetic analysis was correct in the prediction of the fetal sex in seven cases; in two cases the prediction was questionable, and in two cases it was in error. The conclusion is that the use of trophoblast cells for fetal cytogenetic studies is not practical because of (1) difficulties in culturing the cells, (2) the low mitotic frequency of the cells in culture, and (3) contamination of the culture with maternal fibroblasts.

A modified technique is described for obtaining extended metaphase chromosomes (600 bands) from direct preparations of chorionic villi, allowing rapid detection of subtle structural aberrations with minimal risk of maternal-cell contamination. Analysis of multiple individually processed villus fragments may be helpful in recognizing mosaicism confined to chorionic villi.

Tuberculostearic acid [(R)-10-methyloctadecanoic acid (TBSA)] is a structural component of mycobacteria, and its detection in appropriate clinical specimens has potential application as a rapid screening method for the presence of Mycobacterium tuberculosis and other mycobacteria. We used the highly sensitive technique of gas chromatography-mass spectrometry (GC-MS) combined with selected ion monitoring (SIM) of m/e 312 and m/e 167 for detecting TBSA in 405 clinical sputum specimens collected in Hong Kong, where tuberculosis is still common. TBSA was detected in 39 M. tuberculosis smear-positive, culture-positive specimens; 63 of 66 smear-negative, culture-positive specimens; and 1 of 300 smear-negative, culture-negative specimens. Thus, for screening of sputa from individuals with suspected pulmonary tuberculosis, the detection of TBSA by GC-MS/SIM is highly specific and more sensitive than conventional microscopy and more rapid but slightly less sensitive than conventional culture methods.

Total, free and esterified cholesterol were measured in the plasma, high density lipoproteins, low density lipoproteins, and total cholesterol in the very low density lipoproteins of 141 pairs of adult white male twins. Free cholesterol in plasma, high density lipoproteins and low density lipoproteins had significant genetic variance. Esterified cholesterol had greater total variance in dizygotic than monozygotic twins, interpreted as evidence for greater environmental influences on the two types of twins. After bias due to unequal environmental effects on the two types of twins was removed, there was no significant genetic variance for any esterified fraction of cholesterol.

Tuberculostearic acid, a structural component of Mycobacterium tuberculosis, was identified by gas chromatography/mass spectrometry with selected ion monitoring in cerebrospinal fluid (CSF) from 13 patients with proven and 8 out of 9 patients with suspected tuberculous meningitis; the negative result was in a patient whose symptoms and CSF abnormalities may have been due to systemic lupus erythematosus. Tuberculostearic acid was found in the CSF of only 1 patient out of 87 with non-tuberculous meningitis or non-infectious disorders; the single false-positive result was probably caused by intrathecal treatment with amikacin. Other aminoglycosides and antituberculous drugs did not interfere with the assay. In serial CSF samples from patients with tuberculous meningitis tuberculostearic acid was still present after 8 months of continuous supervised chemotherapy. Detection of tuberculostearic acid in CSF is a rapid, sensitive, and specific test for tuberculous meningitis, and can be used for retrospective diagnosis in patients who have been started on therapy.

One metabolite of haloperidol present in plasma is "reduced haloperidol." This study demonstrates that human red blood cells are capable of converting haloperidol to reduced haloperidol in vitro. The reductase involved requires NADPH, as does haloperidol (ketone) reductase in human liver cytosol.