The Hoesch Test: Bedside Screening for Urinary

CLIN. CHEM. 20/11, 1438-1440 (1974)

Porphobilinogen in Patients with Suspected Porphyria

Joel Lamon,’ Torben K. With, and Allan G. Redeker

A little-known procedure for porphobilinogen screening, stead is usually done in the hospital laboratory. We the Hoesch test, was examined for sensitivity, specifici- propose that the Hoesch test, first described in 1947 ty, and utility as compared to the Watson-Schwartz (3) as the “reversed Ehrlich reaction,” replace the test. The data demonstrate that its sensitivity is similar “conventional Ehrlich reaction” (Watson-Schwartz). to that of the Watson-Schwartz test. The Hoesch test, With the same aldehyde reagent originally described however, is without false-positive reactions secondary by Pr#{246}scher(4), the Hoesch procedure is simpler, to urobilinogen, and its simplicity makes it easier to use and more easily interpretable. We conclude that the faster, and appears to be more specific and more easi- Hoesch test should replace the Watson-Schwartz test ly interpreted than is the Watson-Schwartz test. The for urine porphobilinogen screening in suspected cases advantages of this test appear to have been ignored of acute intermittent and variegate porphyria. until recently (5-7).

AdditIonal Keyphras.s: Watson-Schwartz #{149} “reversed Patients and Methods Ehrlich reaction ‘ ‘ #{149}screening The Hoesch test was examined for specificity and sensitivity by comparison with the modified Watson- Acute intermittent porphyria is an inherited de- Schwartz procedure (3 ) on fresh urine specimens fect of porphyrin biosynthesis characterized by an from persons including patients with alcoholic liver excess of porphyrin precursors in the urine. Although disease, hepatitis, porphyria (acute intermittent por- clinical manifestations may vary, the cardinal symp- phyria and variegate porphyria), normals, and nor- tom is abdominal pain. Neuropathy, seizures, coma, mals treated with phenazopyridine HC1 (Pyridium). psychoses, and electrolyte disturbances may occur. Photosensitivity is not a feature of acute intermittent Urobilinogen was measured in parallel with these two porphyrin. An indistinguishable acute illness may tests, as it has been a well-documented cause of falsely positive Watson-Schwartz tests. Patients treated occur during the course of variegate porphyria and with Pyridium were included because use of this drug with hereditary coproporphyria. Diagnosis of acute intermittent porphyria and the has been reported as a source of potential falsely positive reactions. In addition, quantitative porphobili- acute attack equivalents of variegate porphyria and nogen determinations were compared to the qualita- hereditary coproporphyria is based on the demon- stration of increased excretion of porphobilinogen in tive Hoesch test results. These screening test procedures (Watson- the urine. Subsequent studies of the patterns of urine and fecal porphyrin excretion are necessary to define Schwartz and Hoesch) are outlined in Table 1. We have found that the Ehrlich’s reagent used in the the type of porphyria (acute intermittent porphyria, Hoesch test can be stored for at least nine months in variegate porphyria, or hereditary coproporphyria). The test for urinary porphobilinogen customarily a clear-glass container on the shelf without loss of ac- tivity. used in this setting is that developed by Watson and Schwartz in 1941 and modified in 1964 (1, 2). Al- Results though termed a screening test, it is a multistep pro- Specificity of the Hoesch Test cedure, rarely performed on the clinical ward but in- The Watson-Schwartz and Hoesch procedures Department of Internal Medicine, University of Southern Cali- gave almost identical results (Table 2) for normal fornia School of Medicine, Los Angeles, Calif. 90033; and the De- persons, normal persons on Pyridium therapy, and partment of Chemical Pathology, Svendborg Hospital, 5700 for documented cases of acute intermittent porphyria Svendborg, Denmark. 1 Present address: Department of Internal Medicine, Parkland and variegate porphyria. Two cases of the liver-dis- Memorial Hospital, Dallas, Tex. 75235 ease group, specifically two with positive urobilino- Address communications to J. L. at: Dept. of Internal Medicine, gen tests, produced positive interpretations with the University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Tex. 75235. Watson-Schwartz procedure, while the Hoesch test Received June 28, 1974; accepted Aug. 26, 1974. was unreactive.

1438 CLINICALCHEMISTRY. Vol. 20, No. 11, 1974 Table 1. The Urine Screening Tests for Table 2. Results of Ehrlich Reaction Porphobilinogen of Watson and Schwartz () Procedures in Urine and of Hoesch (3) Modified Watson-Schwartz Hoesch Uro- Watson- bilinogen Schwartz () Hoesch (3) Ehriich’s 0.7 g of p.dimethyl 20 g p-dimethyl- +INo. +/No. +/NO. “modified” aminobenzaldehyde, aminobenzal. subjects tested tested tested reagent 150 m of concd HCI, dehyde diluted to Normals (19) 0/19 0/19 0/19 and 100 ml of water 1000 ml with HCI, Liver diseases (54) 21/54 2/54k Q/54 6 mol/liter (cirrhosis and hepatitis) Procedure Equal volumes of urine 2-3 ml of this Porphyria (7) (acute inter- 2/7 7/7 7/7 and modified Ehrlich’s Ehrlich’s reagent mittent porphyria and reagent. To this, an to which is added variegate porphyria) equal volume (i.e., 2 x 2 dropsof fresh Pyridium therapy (4) 2/4 0/4 0/4 1 volume) of a satu- u ri ne.a U Numbers in parentheses are number of persons tested. rated solution of so- b Two positive tests are from the uroblllnogen#{149}posltlvesam- dium acetate.A few pies. ml of chloroform is added and the mixture is thoroughly agitated. Table 3. Relationship of the Hoesch Test to Positive Cherry-red color in the Instantaneous Quantity of Porphobulinogen Present; by the test aqueous phase of the cherry-red color, Method of Mauzerall and Granick ($) tube. If the aqueous present initially at Porphoblilnogen, phase appears posi- the top of the mg/liter Ho.sch test tive, the accurate solution but 42.0 Strongly + screening requires ex- throughout the 20.4 Strongly + traction of the cherry. tube on brief 5.5 + red aqueous phase agitation. 2.2 with butanol. If color persists in the aqueous phase after this second procedure, it indicates the presence of porphobilino. considered in the differential diagnosis of painful ab- gen. dominal disorders and certain neuropsychiatric dis-

a We have modified this procedure to 1 ml of reagent plus orders. The availability of a quick, reliable screening 1-2 drops of fresh urine. procedure is invaluable when the possibility of porphyria is considered. The Watson-Schwartz test has served well as a screening procedure for more than 30 years; however, its use on clinical wards has been re- stricted by (a ) the number of reagents required, (b) One of us (T. K. W.) has screened 50 cases of time for test performance, and (c ) lack of expertise in “acute abdominal” problems, resolved with diagnoses proper performance and interpretation of the test re- other than porphyria-all with a negative Hoesch sults. test. Doss (8 ) further reported negative Hoesch tests The Hoesch procedure has also been available for in all of 300 different patients with chronic liver dis- almost 30 years, and it is interesting to note the com- orders, porphyria cutanea tarda, lead poisoning, and parative evolution of these two procedures. Both polyneuritis. evolved from the strong red reaction produced by acid solution of p- dimethylaminobenzaldehyde with Sensitivity of the Hoesch Test certain urines, originally described by Ehrlich in During porphyric attacks (acute intermittent por- 1900. It was not until 1931 that the potential signifi- phyria), the porphobilinogen concentration in serum cance of porphobilinogen in urine was appreciated will be 10 mg/liter or greater (9). Thus, it is impor- (10-12 ). In the previous years, interest in the clinical tant that a test be unequivocally positive at these di- significance of urobilinogen and its detection in urine agnostic concentrations of porphobilinogen, but not was the major importance of the Ehrlich reagent so sensitive as to detect physiological concentrations (13-18). Hoesch used Ehrlich’s original reagent of porphobilinogen. Table 3 demonstrates that at low (Table 1). Thus, the essential difference between the concentrations, not consistent with an acute attack Watson-Schwartz and Hoesch procedures is that the (2.2 mg/liter), the Hoesch test is nonreactive, and at former attempts to fully develop and then extract the clinically significant concentrations, it is strongly urobilinogen-attributable color, while the Hoesch positive. discovery of the inverse Ehrlich’s reaction (i.e., of maintaining an acid solution by adding a small urine Discussion volume to a relatively large reagent volume) eliminat- Although they are rare diseases, both acute inter- ed the problem of urobilinogen reaction. mittent porphyria and variegate porphyria are often Our data confirm the original observation of

CLINICALCI-IEMISTRY, Vol. 20, No. 11, 1974 1439 Hoesch that the inverse Ehrlich reaction is specific 3. Hoesch, K., Uber die Auswertung der Urobilinogenurie and die for porphobilinogen (Table 2). Falsely negative reac- umgekehrte Urobilinogenreaktion. Deut. Med. Wochenschr. 72, 704 (1947). tions have seldom been a problem with the Watson- 4. Pr#{246}scher,F., Zur Kenntness der Ehrlich#{225}chenDimethylamido- Schwartz test, and the same appears to be true for benzaldehyd-Reaktion. Hoppe-Seyler’s Z. Physiol. Chem. 31, 520 the Hoesch test. With certain indicator compounds (1900). such as Pyridium, in the strongly acid Ehrlich’s re- 5. With, T. K., Screening tests for porphyria. Lancet ii, 1187 (1970). agent color will commonly form that is not character- 6. With, T. K., Screening tests for porphyria. Lancet i, 240 (1971). istic of either urobilinogen or porphobilinogen. Other 7. With, T. K., Simple and rapid screening for acute porphyria- possible sources of falsely positive Ehrlich reactions Porphobilistix’ and Hoesch test. S. Afr. Med. I. 45 (special issue), were not examined (19, 20), nor were potential inhib- 229 (1971). itors of this reaction tested with the Hoesch proce- 8. Doss, M., Erwiderung auf die ‘comments on paper, zwei Suchteste f#{252}rPorphyrien’. Z. Kim. Chem. KIm. Biochem. 11, 130 dure (21 ). The Hoesch test does not detect the low (1973). concentrations of porphobilinogen that are present in 9. Stein, J. A., and Tschudy, D. P., Acute intermittent porphyria. normal urine (Table 3). The value of the Hoesch test Medicine (Baltimore) 49, 1 (1970). lies in screening for suspected acute attacks. In such 10. Sachs, P., Em Fall von akuter Porphyrie mit hochgradiger cases the porphobilinogen concentration in the urine muskel Atrophie. Kim. Wochenschr. 10, 1123 (1931). 11. Waldenstr#{246}m,J., Some observations on acute porphyria and is typically 10 mg/liter. When such an amount is other conditions with a change in the excretion of porphyrins. Acta present, the positive Hoesch test is unequivocal Med. Scand. 83, 281 (1934). (Table 3). The dilemma of interpreting “weakly posi- 12. Waldenstr#{246}m,J., and Wahlquist, B., Studien Uber die Entate- tive” tests will only arise if the test is extended to a hung der roten Harnpigmente (Uroporphyrin und Porphobilen) bei der akuten Porphyrie aus ihrer farblosen Vorstufe (porphobili- search for carriers. We have encountered several nogen). Hoppe-Seyler’s Z. Physiol. Chem. 260, 189 (1939). cases of “suspicious” Watson-Schwartz tests that 13. Herter, C. A., On a relation between skate! and the dimethyla- have been negative by quantitative methods. With minobenzaldehyde (para ) reaction of the urine. J. Biol. Chem. 1, the Hoesch test the presence of porphobilinogen in 251 (1905). urine can be quickly and accurately identified and 14. Neubauer, 0., Ueber die neue Ehrlichschen Reaktion met Di- methylaminobenzaldehyd. Ges. Morphol. Physiol. Munchen 19, further investigative procedures instituted to identify 32 (1903). the type of porphyria. 15. Wallace, G. B., and Diamond, J. S., The significance of urobili- We suggest that the Hoesch test replace the Wat- nogen in the urine as a test for liver function. Arch. Intern. Med. 35, 698 (1925). son-Schwartz test. In no way is it less effective, and 16. Terwen, A. J. L., Uber em neues Verfahren zur quantitativen in several it is better. It is not our intention to add Urobilin-Bestimmung in Ham and Stuhl. Deut. Arch. Kim. Med. another test to the diagnostic armamentarium of 149, 72 (1925). medicine, rather to improve the methodology for effi- 17. Watson, C. J., An improved method for the quantitative esti- mation of urobilinogen in urine and feces. Amer. J. Clin. Pathol. 6, cient diagnosis. We feel sufficient criteria have been 458 (1936). met to warrant replacing the Watson-Schwartz test 18. Naumann, H. N., A study of Ehrlich’s test for urobilinogen with the more efficacious Hoesch test. and Schlesinger’s reaction for urobilin. Biochem. J. 30, 347 (1936). 19. Reio, L., and Wetterberg, L., False porphobilinogen reactions The technical assistance of Mrs. Patricia Hoyte is greatly appre- in the urine of mental patients ,J. Amer. Med. Ass. 207, 148 (1969). ciated. 20. McEwen, J., and Paterson, C., Drugs and false-positive screen- References ing tests for porphyria. Brit. Med. J. 1, 421 (1972). 21. Taddeini, L., Kay, I. T., and Watson, C. J., Inhibition of the 1. Watson, C. J., and Schwartz, S., A simple test for urinary por- Ehrlich’s reaction of porphobilinogen by indican and related com- phobilinogen. Proc. Soc. Exp. Biol. Med. 47, 393 (1941). pounds. Clin. Chim. Acta 7, 890 (1962). 2. Watson, C. J., Taddeini, L., and Bossenmaier, I., Present status 22. Mauzerall, D., and Granick, S., The occurrence and determina- of the Ehrlich aldehyde reaction for urinary porphobilinogen. J. tion of delta-aminolevulinic acid and porphobilinogen in urine. J. Amer. Med. Ass. 190, 501 (1964). Biol. Chem. 219, 435 (1956).

1440 CLINICALCHEMISTRY, Vol. 20, No. 11, 1974