O.95x + 3.90 ... The interassay CVs were: toluene 9.2%, benzene 10.2%. The .... Ann. Rev Immunol 1985;3:477-500. 2. Stahl RE, Kien AF, Dubin R, Marmor M, ...
Technical Briefs (-300 words text) summarize findings that are of interest to a relatively limited audience. Readera desiring fuller details may obtain them by writing directly to the author(s) at the address given or, if this is impossible for any reason, to the EditOrial office of this journal.
Thin-Layer Chromatography of Homovanillic AcId with Use of Toluene Instead of Benzene, Jerome M. Feldman and Charles Castleberry (Durham Veterans Administration Medical Center and Dept. of Med., Duke University Medical Center, Durham, NC 27710) For 16 years we have determined the urinary excretion of 4-hydroxy-3-methoxyphenylacetic acid (homovanilhic acid, HVA) by a thin-layer chromatographic (‘PLC) method in which the urine extracts are separated on silica gel G plates with the solvent system benzene:glacial acetic acid:water (2:3:1 by vol). The only modification we make in the published method (1) is to analyze each sample in duplicate and to air-dry the plates and develop them a second time before they are sprayed with Folin’s phenol reagent. In recent years excessive exposure to benzene has been associated with development of leukemia, and permissible exposure to it has been recently revised (2). We studied 58 urine samples from healthy subjects, patients with various illnesses, and patients with dopainine overproduction from pheochromocytomas, neuroblastomas, or carcinoid tumors to see if toluene could be substituted for benzene in the mobile phase of the TLC (3). The data were analyzed by the paired f-test and by regression analysis (4). 140
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Fig. 1. HVA measurements in 58 urine samples compared when the thin-layerchromatographywas done with the mole phase contalning toluene or benzene Thecorretationcoef,cientwas 1.00; the equationof theregreson line is y = O.95x + 3.90
1. Sankoff I, Sourkes U. Determination by thin-layer chromatography of urinary homovanillic acid in normal and disease states. Can J Biochem Physiol 1963;41;1381-88. 2. Occupational exposure to benzene: final rule. Fed Reg 1987;52:34480-578. 3, Feldman JM. Increased dopaniine production in patients with carcinoid tumors. Metabolism 1985;34:255-60. 4. Zar JH. Biostatistical analysis, Englewood Cliffs, NJ; Prentice Hall, Inc., 1974.
Effect of Storage pH on Precipitation of Albumin from Urine from Diabetics, J. C. Townsend, P. J. Blair, and A. R. W. Forrest (Dept. of Clinical Chemistry, Royal Hospital,
Sheffield
SlO 2JF, U.K.)
subjects.
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References
One ofus has reported (1, 2) that albumin may precipitate from urine samples stored at -20 #{176}C. This observation, made on urine samples from normal subjects, has not been endorsed by others (3) investigating urine from diabetic
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the mean R1 for HVA was less with toluene (0.59), the HVA was cleanly separated from surrounding compounds in both systems. The interassay CVs were: toluene 9.2%, benzene 10.2%. The corresponding intra-assay CVs were 8.3% and 8.6%. The measured range of excretion in normal subjects was similar with both solvents (5.5 to 65.9 nmol/24 h). Figure 1 shows that the mean ± SEM of the toluene sample expressed as a percentofthe paired benzene sample was 101 ± 1.7%. There was no significant difference by the paired t-test. The correlation coefficient using the two mobile phases was 1.00 and the regression equation was y = 0.95x + 3.90, where y was with benzene. Evidently toluene can be successfully substituted for benzene in this analytical method. Although
(0.44) than with benzene
We investigated whether this phenomenon occurs in the urine ofdiabetic subjects and is influenced by urine composition, and whether adjusting the urinary pH overcomes the problem. We collected untimed mid-stream urine samples from 39 unselected diabetic patients attending an afternoon clinic. We tested or measured, for each sample, glucose (BM-Test-7 strips; BCL, London, U.K.), urinary pH (pH meter 130, Corning, Haistead, Essex, U.K.), osmolality (Advanced Instruments Inc., Needhain Heights, MA 02194), and albumin (radioimmunoassay; Pharmacia, Uppsala, Sweden). In each case we divided the sample and adjusted one aliquot to pH 7 with 1 mol/L sodium hydroxide or hydrochlonc acid. Portions ofthese paired samples were then assayed within 3 h of voiding. The rest were assayed again after eight weeks ofsthrage at -20 #{176}C. The portion stored without prior pH adjustment was assayed twice, first at its unadjustCLINICAL
CHEMISTRY,
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ed pH and then after adjustment to pH 7. All samples were centrifuged (1000 x g, 10 mm) before analysis. Albumin concentrations ranged from 1.3 to 41.8 mg/L in 33 samples, the remaining six exceeding the upper limit of the method (80 mgIL). We compared the results of different sample treatments by paired t-test and found a significant decrease only for samples stored at -20 #{176}C without pH adjustment (P 4 gIL. Ten subjects had increased IgA at all three testings; 10 had increased IgA at the second and third testing but not at the first one; and five had increased IgA at the third testing only. Thus, after one year offollow-up, 25 subjects (31.2%) had an increased IgA. The existence of this anomaly was not correlated with a particular risk factor. The average number of helper (CD4) lymphocytes of subjecta with increased IgA during the study period was 410 per microliter at the first testing and 170 per microliter at the third; the average CD4 lymphocytes count of subjects with normal IgA concentration at all three testings was 590 per microliter at the first examination and 420 per microliter one year later, significantly different from the first group of subjects (P 6 gIL at the third testing had a mean of 1 12 CD4 per microliter at the last testing; the subjects with an IgA concentration between 4 and 5 g/L at the first testing and at
