MATERNAL ALPHA-FETOPROTEIN AND FETAL EXOMPHALOS

303 SCREENING TEST FOR INTERMITTENT ACUTE PORPHYRIA were for of intermittent acute porphyria (I.A.P.).’ However, the limitations of this test, particularly in ascertaining individuals with latent or quiescent disease, are well known.2 The demonstration of the primary biochemical-genetic defect in I.A.P., deficient activity of the enzyme uroporphyrinogen-I-synthetase (uro.-s.),3 established a means for making the diagnosis in virtually all affected individuals. We wish to report an adaptation of a quantitative assay for erythrocyte uro.-s.,4 suitable for the rapid screening of large populations for the presence of I.A.P. Blood is spotted dropwise on Schleicher and Schuell No. 903 filter-paper and dried. A -L-inch 4 disc is punched from the centre of a drop and placed in a tube with 2.0 ml reaction solution (0-11 mmol/1 porphobilinogen in 45 mmol/1 "tris"-(HCl), pH 8-2). After incubation for 3.5h at 37°C in the dark, with occasional shaking, the reaction is terminated with 2.0 ml 25% trichloracetic acid. After centrifugation at room temperature, the supernatant is removed to a lOx75 mm glass tube. The tube is inserted through a piece of black paper into a wire testtube rack, and illuminated in the dark with a long-wavelength ultraviolet hand lamp situated below the paper and at the side of the tube. The fluorescence of the porphyrin products is observed visually through the mouth of the tube above the

Sm,—Urinary porphobilinogen determinations

many years essential for the

diagnosis

paper. Blood from normal subjects, whether spotted directly or anticoagulated with any of the commonly used agents, exhibits bright-pink fluorescence. Fluorescence intensity remains unchanged if the blood spots are stored at either 4C for up to 9 days or 250C for 1 or 2 days. Spots stored at -20°C are stable for at least a month. Fluorescence in samples from five

patients with I.A.P. is of a different colour (violet) and intensity (much reduced), making discrimination obvious. Fluorescence in samples of severely anaemic blood is also reduced, and may give falsely positive results. However, blood-haemoglobin concentration may be determined accurately with these same spots by eluting the haemoglobin in water and measuring the optical density in the Soret region against appropriate standards. Difficulty from another source of false-positive tests-variation in enzyme activity creating an overlap zone of normal and porphyric results-is anticipated but has as yet not been a confounding problem. We are presently using this method to screen residents of several inpatient psychiatric facilities in Massachusetts. A full description of this method is in preparation. Channing Laboratory, Boston City Hospital, Boston, Massachusetts 02118, U.S.A. Department of Medicine, Harvard Medical School, Boston Dartmouth Medical School, Hanover, New Hampshire.

PETER V. TISHLER

DANIEL

J. KNIGHTON

HENRY M. SCHUMAKER

AMNIOTIC FLUID OR MATERNAL URINE?

SIR,-Quite early in our programme of amniocentesis, which started in 1971, we recognised the risk of inadvertently tapping the maternal urine instead of amniotic fluid, especially in cases of anterior placenta requiring a transabdominal lowersegment approach. We have been using a simple spot test to differentiate maternal urine from amniotic fluid. The fluid withdrawn at the time of amniocentesis is tested with a ’Lab1

Watson, C. J., Taddeini, L., Bossenmaier, I. J.

Am. med. Ass.

1964, 190,

501. 2

Tschudy, D. P., Valsamis, M., Magnussen, C. R. Ann. intern Med. 1975, 83,

3

Strand, L. J., Meyer, U. A., Felsher, B. F., Redeker, A. G., Marver, H. S. J clin. Invest. 1972, 51, 2530. Magnussen, C. R., Levine, J. B., Doherty, J. M., Cheesman, J. O., Tschudy, D. P. Blood, 1974, 44, 857.

851.

4

strip (Ames). The manufacturers state that this detect albumin in concentrations of 5-20 mg/dl urine in "trace" amounts). The test is more sensitive to albu-

stix’ reagent

strip (i.e.,

can

min than to globulin, Bence-Jones protein, or mucuprotein. The sensitivity to glucose is about 0-1% (100 mg/dl) although it is influenced by inhibiting substances, specific gravity, temperature, and pH of the urine. In the absence of diabetes mellitus or renal disease, maternal urine contains very little, if any, glucose or protein, whereas amniotic fluid has a lot of both. We have had three instances where maternal urine was obtained at amniocentesis. The two from our centre were detected at once using the reagent strip and amniocentesis was repeated immediately. One specimen, received from another centre, was from a 26-year-old woman with a previous history of delivery of an anencephalic fetus. Amniocentesis was done at 16 weeks’ gestation. No a-fetoprotein (A.F.P.) was detected in this specimen. However, no glucose or protein was recorded on strip testing, and the fluid contained 14.4 mg creatinine/dl. We concluded that this was a sample of maternal urine, and amniocentesis was repeated at 18 weeks’ gestation when the amniotic-fluid A.F.P. was 9.1mg/dl. The pregnancy was terminated and the hydrocephalic fetus with spina bifida, omphalocele, and bilateral club feet was delivered. The use of reagent strips to differentiate between amniotic fluid and urine is quick, simple and accurate in this situation in the absence of maternal diabetes or renal disease, and by this means we have virtually eliminated the possibility of falsenegative results due to maternal urine. Amniotic Fluid Study Office, Department of Obstetrics and Gynæcology, University of Toronto, Toronto General Hospital, Toronto, Ontario M5G 1L7, Canada.

B. B. K. PIRANI T. A. DORAN R. J. BENZIE

MATERNAL ALPHA-FETOPROTEIN AND FETAL EXOMPHALOS

SIR,-Raised amniotic-fluid alpha-fetoprotein (A.F.P.) concentrations have been reported in association with fetal exomphalos.’We describe here a case in which the maternal

abnormally high. 41-year-old woman was admitted because of uterine contractions and slight bleeding at 25 weeks of pregnancy. Fetal haemoglobin could not be detected in the bleeding. Her serum-A.F.P. measured by radioimmunoassay3 (1000 ng/ml and 780 ng/ml a week later) was well above the upper limit of normal for 26 weeks’ gestation.4 Bleeding and contractions ceased during isoxuprine treatment and the patient went

serum-A.F.P. was

A

home. At 27 weeks she returned in labour. The fetal heart action was normal at first, but stopped just before birth. A 1000 g infant was born with a large ruptured exomphalos, truncuc arteriosus in the heart, small polycystic kidneys, and urethral and a high-rectal atresia. No neural-tube defect was present. No specific placental cause for uterine bleeding could be demonstrated. Raised maternal serum-A.F.P. levels have been found in association with transplacental leakage, intrauterine death,4and fetal neural-tube defects6but we found no neural-tube defect, and the raised A.F.P. levels were detected well before fetal death. Thus, the most likely reason for raised maternal A.F.P. levels in this case is fetal exomphalos, and, perhaps, the other associated malformations. At least 50% of fetuses with exomphalos have other malformations, including anencephaly in 20% of cases in Britain.8 1. De Bruijn, H. W. A., Huisjes, H. J. Lancet, 1975, i, 525. 2. Nevin, N. C., Armstrong, M. J. Br. J. Obstet. Gynœc. 1975, 82, 826. 3. Ruoslahti, E., Seppälä, M. Int. J. Cancer, 1971, 8, 374. 4. Seppälä, M. Ann. N.Y. Acad. Sci. 1975, 259, 59. 5. Seppälä, M., Ruoslahti, E. Lancet, 1972, 1, 375. 6. Brock, D. J. H., Scrimgeour, J. B., Bolton, A. E., Wald, N., Peto, R., Barker, S. ibid. 1975, ii, 195. 7. Leighton, P. C., Gordon, Y. B., Kitau, M. J., Leek, R. E., Chard, T. ibid. 1975, ii, 1012. 8. McKeown, T., MacMahon, B., Record, R. G. Am. J. hum. Genet. 1953, 5, 169.

304 Not many parents would want to continue a pregnancy even though the anticipated malformation was "minor", and since mild and severe malformations cannot be distinguished antenatally and since raised levels are usually related to fetal defects other than exomphalos,’-’ termination of these pregnancies seems justified provided that false positives are

F.D.P. was abnormal in only 75%. We agree with Purdie et al. that amniotic-fluid F.D.P. is a valuable but not completely consistent marker in the prenatal detection of N.T.D., especial before the 24th week of gestation. Further evaluation of the accuracy and specificity of this method is warranted.

excluded.

some

This study was carried out under a contract with the Association of the Finnish Life Insurance Companies.

Departments of Obstetrics and Animal Research, Nassau County Medical Center, East Meadow, N.Y. 11554, and Department of Medicine,

Department II of Obstetrics and Gynæcology, University Central Hospital, 00290 Helsinki 29, Finland; Childrens’ Hospital, University of Helsinki; and Department of Serology and Bacteriology, University of Helsinki.

M. SEPPÄLÄ O. KARJALAINEN J. RAPOLA J. LINDGREN

F.D.P. IN AMNIOTIC FLUID AS MARKER FOR NEURAL-TUBE DEFECTS

SIR,-Purdie

et

al.1 suggest that the level of fibrin/fi-

brinogen degradation products (F.D.P.) in amniotic fluid may be of value as a protein marker of fetal neural-tube defects (N.T.D.). We have measured F.D.P. in 53 amniotic fluids from normal pregnancies (10-40 weeks’ gestation) and in 17 amniotic fluids from sixteen cases of N.T.D. F.D.P. were measured by the automated haemagglutination-inhibition assay.2 The results are presented in the accompanying figure.

Weeks of Gestotion F.D.P. levels in normal and N.T.D. amniotic fluid.

normal amniotic-fluid F.D.P. is in the same that described by Purdie et al., and we agree that F.D.P. levels in normal amniotic fluid seem to be independent of gestational age. The mean (±S.D.) value for 10-24 weeks is 2.37 + 1.59 and for 25-40 weeks it is 2.12 + 1.66 p.glml fibrinogen equivalents. Before the 24th week F.D.P. levels in 4 of 5 amniotic fluids of anencephalic pregnancies and 2 of 3 spina-bifida pregnancies were significantly raised (above mean + 2s.D.). After the 24th week only 1 of the 9 N.T.D. amniotic fluids showed significant elevation. Since selective abortion should be done before 24 weeks this lack of diagnostic accuracy in late gestation should not detract from the test’s potential value in early pregnancy. All samples tested for F.D.P. were also evaluated for a-fetoprotein (x-F.P.). No correlation between a-F.P. and F.D.P. was observed in normal fluids. In N.T.D. fluids M-F.p. was significantly raised in all samples before 24 weeks,3 whereas the Our

mean

general range

as

Howie, P. W., Edgar, W., Forbes, C. D., Prentice, C. R. M. Lancet, 1975, i, 1013. 2. Merskey, C., Johnson, A. J., Lalezan, P.J. clin. Invest. 1972, 51, 903. 3. Weiss, R. R., Macri, J. N., Elligers, K. W., Princler, G. L., McIntire, K. R., Waldermann, T. A. Obstet. Gynec. (in the press). 1. Purdie, D. W.,

We thank Dr D. J. H. Brock and Dr R. Harris for supplying us with of the N.T.D.-related amniotic fluids.

Albert Einstein College of Medicine of Yeshiva University, Bronx, N.Y. 10461, U.S.A.

R. R. WEISS

J. N. MACRI C. MERSKEY

METRONIDAZOLE AND IRON IN CANCER THERAPY

SIR,-Without wishing to decry the emphasis Dr Adams and his colleagues (Jan. 24, p. 186) have placed on their important studies with Ro-07-0582, their two short paragraphs on metronidazole do not reflect the potential of this drug in radiotherapy and in cancer therapy generally. In Canada clinical evidence for the value of metronidazole as a radiosensitiser comes from a study in patients with supratentorial glioblastomas by Urtasun et al.’ and a preliminary trial in carcinoma of the bladder is being started at the Churchill Hospital, Oxford. Furthermore, metronidazole also exhibits a chemotherapeutic action on hypoxic cells in vivo in the absence of radiation. Administration of metronidazole to tumour-bearing mice for several hours, before but not immediately before, radiation treatment, lowers the dose required to control 50% of the tumours.23 This is attributed to a cytotoxic effect of the drug on the hypoxic cells present. The very fact that these cells are hypoxic because they are relatively distant from a blood-vessel also means that they will be less likely to be affected by standard chemotherapeutic agents given systemically. Thus the concomitant administration of an agent, such as ’metronidazole, which is selective for hypoxic cells, may result in an increase in the success-rate of chemotherapy. This cytocidal action of metronidazole is supported by studies with mammalian cells in spheroid culture and in ascites tumour cells incubated in vitro,2-4 where no toxicity was observed whenoxygen was present. This accords with the action of the drug being due to a short-lived toxic species, formed either biochemically or by the action of radiation. This toxin is not formed, or is inactivated, when oxygen is present, so normal tissues are not affected.5 A similar mechanism has been proposed for the drug’s selective cytocidal action on anaerobic rather than aerobic microorganisms.6 Indeed, it was this property and its favourable pharmacological and toxicological properties, together with the fact that its structure contained a nitro group that provided the initial stimulus for studying the drug’s radiosensitising action.’ A wide variety of compounds had been studied for sensitising activity.’ The "electron affinity" concept, per se, on which basis many compounds have been chosen for screening, is too broad to be clinically successful. Several compounds of high-electron affimty have been studied in vitro but have proved disappointing or have been unsuitable for study in vivo. The electron affinity of metronidazole is very low relative to other compounds studied,5 but this means that it is relatively favoured pharmacologically and toxicologically and that higher doses may be tolerated clinically. As in all forms of chemotherapy, a balance must be struck. 1.

Urtasun, R. C., Chapman, J. D., Band, P., Rabin, H., Fryer, C., Sturmwind. J. Radiology, 1975, 117, 129. 2. Foster, J. L., Willson, R. L. Proc. IXth int. Congr. Chemother. (in the press 3. Foster, J. L., Conroy, P. J., Searle, A. J., Willson, R. L. Br.J. Radiol (in the

press).

4. Sutherland, R. M. Cancer Res. 5. Willson, R. L., Cramp, W. A.,

1974, 34, 3501. R. M. J. Int. J. Radiat. Biol. 1974, 26,

Ings,

557. 6.

Ings, R. M. J., McFadzean, J. A., Ormerod, W. E. Biochem. Pharmac. 1974, 23, 1421. 7. Foster, J. L., Willson, R. L. Br. J. Radiol. 1973, 46, 234. 8. Adams, G. E. Br. med. Bull. 1973, 29, 48.