9, 1986 1777 activity as measured by the Abbott ER-EIA is similar to that of estrogen binding activity as measured by the DCC method. Thus, the ER-EIA method ...
activity as measured by the Abbott ER-EIA is similar to that of estrogen binding activity as measured by the DCC method. Thus, the ER-EIA method is no substitute for careful handling of the tissue before the assay. We thank Carol Hopkins Clayton, C.M.T., for help in manuscript preparation, and Edward Ashwood, M.D., for advice regarding statistical analysis.
References 1. Newsome JF, Avis FP, Hammond JE, Sherwood S. Sampling procedures in estrogen receptor determinations. Ann Surg 1981;193:549-53. 2. Hasson J, Luhan PA, Kohl MW. Comparison of estrogen receptor levels in breast cancer samples from mastectomy and frozen section specimens. Cancer 1981;47:138-9. 3. Rosenthal LI. Discrepant estrogen receptor protein levels according to surgical technique. Am J Surg 1979;138:680-1. 4. Zachariah NY, Chakmakjian ZH. Stability of estrogen receptor concentrations in human uterus tissue. Clin Chem 1983;29:1070-2. 5. Korenman SG. Radioligand binding assay of specific estrogen using a soluble uterine macromolecule. J Clin Endocrinol Metab 1968;28:127-30. 6. Abbott Laboratories Instruction Manual for Eus.4 estrogen receptor assay, Abbott Laboratories, Chicago, IL, 1984. 7. Cornbleet PJ, Gochman N. Incorrect least-squares regression coefficients in method-comparison analysis. Clin Chem 1979;432-8. 8. Press MF, Greene GL. An immunocytochemical method for
demonstrating estrogen receptor in human uterus using monoclonal antibodies to human estrophilin. Lab Invest 1984;50:480-6. 9. King WJ, DeSombre ER, Jensen EV. Comparison of immunocytochemical and steroid-binding assays in human breast tumors. Cancer Res 1985;45:293-304. 10. Katzellenbogen JA, Johnson JH, Carlson KE. Studies on the uterine cytoplasmic estrogen receptor binding. Biochem 1973;12:4092-9. 11. Sherman MR, Thazon FB, Miller LK. Estrogen receptor cleavage and plasminogen activation by enzymes in human breast tumor cytosol. Endocrinology 1980;106:1715-27. 12. Skovgaard Poulsen H, Ozello L, King WJ, et al. The use of monoclonal antibodies to estrogen receptors for immunoperoxidase detection of ER in paraffin sections of human breast cancer tissue. J Histochem Cytochem 1985;33:87-92. 13. King WJ, Greene GL Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature (London) 1985;307:745-7. 14. Yeager YL, Joseph JL, Hunt JC, et al. Comparison of estrogen receptor levels determined by the Abbott enzyme immunoassay and by steroid binding assays [Abstract]. Clin Chem 1985;31:1009. 15. Sakai F, Saiz S. Existence of receptors bound to endogenous estradiol in breast cancers of premenopausal and postmenopausal women. Steroids 1976;27:99-.11O. 16. Thorsen T. Occupied and unoccupied oestradiol receptors in nuclei and cytosol from human breast tumors. In: Research on steroids, vol.9. Adlercreutz H, Bulbrook RD, Van der Molen IU, et al., eds. Amsterdam: Excerpts Medica, 1981:18-25.
CLIN. CHEM. 32/9, 1777-1 778 (1986)
Urine DiscolorationafterAcetaminophenOverdose P. M. S. Clark,1 J. D. A. Clark,2 and T. Wheatley2 Three patientswith acetaminophenoverdosewere observed to have dark-brown urine at an early stage of their illness. Subsequently, acute anuric renal failure and hepatic dysfunction developed in all three. p-Aminophenol was identified by chromatographic and colorimetric methods in the urine of
each case and is thoughtto be responsiblefor the discoloration. AddItIonal Keyphrases: p-aminophenol sis
chromatography
enzymatic analy-
Case HIstories Case 1: A 51-year-old woman was admitted 20 h after she had ingested 75 g of acetaniinophen, 320 mg of codeine phosphate, and an unknown quantity of nitrazepam. Darkbrown urine was observed 16 h later, when she was already oliguric. She rapidly developed acute anuric renal failure, (plasma creatinine 337 moI/L) and a moderately increased Departments of’ Clinical Biochemistry and 2Medicine, Addenbrooke’s Hospital, Hills Road, Cambridge, U.K. Received April 7, 1986; accepted June 5, 1986.
(776 UIL) serum alanine aminotransferase (EC 2.6.1.2, ALT, normal range 6000 U/L) and she was observed to have brown urine. Despite full supportive management she developed anuric renal failure (plasma creatinine 908 pmol/L) and hepatic encephalopathy, but after ventilation and hemodialysis she recovered and was diecharged three weeks later. Subsequent follow-up revealed normal renal and hepatic function. Owing to the delay in presentation, none of these patients was treated with N-acetylcysteine. Their serum acetaminophen concentrations on admission were 4.8, 2.0, and 0.3 CLINICAL CHEMISTRY, Vol.32, No. 9, 1986
1777
mmol/L (a high risk of hepatotoxicity being associated with concentrations exceeding 0.3 mmol/L at 12 h after ingestion). Histopathological examination of the kidneys and livers in the first two cases demonstrated necrosis of the proximal tubular epithelium and centrilobular hepatic necrosis.
A
B
C
D
Materials and Methods The urine was analyzed colorimetrically and by thin-layer chromatography. The colorinietric assay (1) we used (Porton Products, Wrexham, U.K.) measures both acetaminophen and p-aminophenol. Any p-aminophenol present can be determined by the second reaction of the assay with o-cresol and ammonia/copper ions without added enzyme. For thinlayer chromatographic analysis of the urine we used silicacoated F plates (Merck, Darmstadt, F.R.G.) and a mobile phase of butanol:acetic acid:water (3:1:1 by vol). Aqueous acetaminophen and p-aminophenol standards were also analyzed.
Results p-Aminophenol was demonstrated in the urine of all three cases by the colorimetric assay (Table 1). Thin-layer chromatographic analysis of the urines (Figure 1, plate viewed at 254 nm) and standards indicated the presence of compounds with the same R values as acetaminophen (0.73) and p-aminophenol (0.69 and 0.83). The latter (B) showed two bands, both turning dark brown when exposed to visible light.
Discussion There are several well-recognised causes of brown urine, including liver disease and hemolytic anemia, where bilirubin and urobiinogen are responsible for the color. The heme derivatives, methemoglobin and myoglobin, may also cause brown urine. Less commonly, brown urine may be due to melanin (oxidized from melanogen in malignant melanoma) and polymerized homogentisic acid (alkaptonuria). In our three cases the urinary discoloration may have been partly due to bile pigments, but the brown urine was considerably darker than expected with hepatic dysfunction and developed in case 1 before biochemical evidence of liver damage.
Table 1. Concentrations of Acetamlnophen (A) and p-Amlnophenol (B) In Serum and Urine of Three Overdose Serum Dayafter
A
Urine
mmoUL
admluten
mmoIlL
Case 1
3 Case 2
3
4.8 0.9
0.13 0.03
2.0
ND
1.4 0.6
ND ND
0.3 0.1
0.03 0.03
-
-
2.2
2.0
-
Case 3 3 ND = not detected.
04 0.1
0.1 ______________
1778 CLINICALCHEMISTRY, Vol. 32, No. 9, 1986
None of the patients had hemolytic anemia, melanoma, or alkaptonuria, and the urinary electrophoretogram showed no myoglobin in the first case. In cases 2 and 3, dipstick testing was positive for bilirubin and urobilinogen when these patients were first noted to have brown urine. Brown urine has also been described in four cases of phenacetin overdose and, although the nature of the brown pigment was uncertain, acetaminophen and its metabolites might have been responsible (2). In contrast, brown urine has not been documented in cases of acetaminophen overdose, although serum samples from such cases may become brown on storage (3). Solutions of p-anunophenol undergo oxidation in neutral or alkaline conditions and may in time polymerize to yield a brown color. Brown et al. (3) showed that the pigmentation of stored serum from overdose patients could be attributed to polymerization of p-aminophenol. We therefore postulate that the brown urine in our three cases of acetaminophen overdose was ascribable to p-aminophenol and its polymerization products. The metabolism of acetaminophen is complex (4,5), and the mechanism for the production of p-aminophenol has yet to be determined.
References
B
A
less sample was appbed
We thank Dr. C. Price and R. S. Campbell for their helpful advice.
Cases B
Fig. 1. Thin-layerchromatogram (viewed at 254 nm) of urine (A, C) from case 1 at different sampleloadings,p-aminophenolsolution (, and urine from case 1 pIusp.aminophenol (D) Cen airow, p-aminophenol;black airow, acetaminophen.Cs fainterbecause
01
ND
1. Hammond PM, Scawen MD, Price CP, et al. Development of an enzyme-based assay for acetaminophen. Anal Biochem 1984;143:152-7. 2. Miller AL, Worsley LR, Chu PK. Brown urine as a clue to phenacetin intoxication. Lancet 1970;ii:1102-4. 3. Brown SS, Campbell RS, Price CP, et al. Collaborative trial of an enzyme-based assay for the determination of paracetamol in plasma. Ann Clin Biochem 1983;20:353-9. 4. Corcoran GB, Mitchell JR, Vaishnav YN, Horning EC. Evidence that acetaminophen and N-hydroxyacetaminophen form a common arylating intermediate, N-acetyl-p-benzoquinoneimine. Mol Pharmacol 1980;18:536-42. 5. Bales JR. Sadler PJ, Nicholson JK, Timbrell JA. Urinary excretion of acetaminophen and its metabolites as studied by proton NMR spectroscopy. Clin Chem 1984;30:1631-6.
