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RADIOIMMUNOASSAY OF ALPHA-FETOPROTEIN IN HUMAN PLASMA
1355
neoplasms. 5 of the patients with
RADIOIMMUNOASSAY OF ALPHAFETOPROTEIN IN HUMAN PLASMA
J.
A. P. CHAYVIALLE
the other 17
A
highly sensitive radioimmunoassay capable of measuring &agr;1-fetoprotein been has developed. A.F.P. was detected in the (A.F.P.) all 105 controls (2·3±1·2 ng. per ml.). of the plasma There was no significant difference between the A.P.P. level of controls and those with histologically proven secondary liver tumours (13 patients) or extrahepatic neoplasms (23 patients). In 17 of the 22 patients (77%) with primary hepatocellular carcinoma, the A.F.P. level was very elevated (2·5 µg. per ml. to 2·6 mg. per ml.), in another 3 patients (14%) it was moderately high (10-25 ng. per ml.), while in 2 of the 22 patients (9%) it
was
within the normal range. Introduction
THE
serum
of the human fetus contains
an
OC1-
globulin called oc-fetoprotein (A.F.P.). Using insensitive immunological methods A.F.P. was detected in 40-80% of the sera of patients with primary hepatocellular carcinoma (P.H.C.c.) but not in controls .2-5 The percentage of positive results was higher when more sensitive techniques were used for the estimation of A.F.P.6A.F.P. has been measured by radioimmunoassay. 7 ,We report here plasma-A.F.P. levels in controls and in patients with P.H.C.C. and liver metastases or extrahepatic malignancies arising from the gut, lungs, thyroid, prostate, or pituitary gland, using a highly sensitive radioimmunoassay (R.I.A.) method. Patients and Methods
Patients We studied 163 patients. There were equal numbers of males and females. 105 patients were hospital controls with no clinical or biochemical evidence of liver disease. The other 58 patients had histologically proven tumours22 were P.H.C.C., 13 liver metastases, and 23 extrahepatic
Pembrey, M. E., Weatherall, D. J., McWade, P. J. clin. Path. 1972, 25, 738. 6. Kleihauer, E., Braun, H., Betke, K. Klin. Wschr. 1957, 35, 637. 7. Weatherall, D. J., Gilles, H. M., Clegg, J. B., Blankson, J. A., Mustafa, D., Boi-Doku, F. S. Ann. trop. Med. Parasit. 1971, 65, 5.
9. 10. 11.
12. 13. 14. 15.
P.H.C.C. were
Europeans,
origin.
The highly purified A.F.P. used as standard and for radioiodination and the sheep anti-human A.F.P. serum were both donated by Dr E. Ruoslahti. A.F.P. was iodinated with iodine-125 (Radiochemicals Centre, Amersham) using the chloramine-T method of Hunter and Greenwood.9 The sheep anti-human A.F.P. serum was titrated by incubating it in serial dilutions (1/5000 to 1/2,560,000) -with 50 pg. 225I-A.F.P. in 005M phosphate buffer, pH 7-5, with 1% horse serum, at 4°C for 6 days. Antibody-bound 12sI-A.F.P. was separated from free 125I-A.F.P. by a double-antibody method’IO using 1/20,000 non-immune sheep serum and a rabbit anti-goat gamma-globulin serum (As 322, Wellcome Reagents) at a final dilution of 1/200. 24 hours later, after adding cold diluent, the tubes were centrifuged at 2000 g at 4°C for 60 minutes. The supernatant was discarded and the radioactivity of the precipitate counted in an automatic gamma spectrometer (Wallac). Standard inhibition curves were obtained by incubating serial amounts of standard A.F.P. (50-3200 pg.) with antiserum at a final dilution of 1/120,000 at 4°C for 2 days. 50 pg. 125I-A.F.P. was then added to each tube and they were incubated for a further 48 hours. Antibody-bound and free 115I-A.F.P. were
separated. Plasma or serum was obtained from patients and stored -30°C. Before assay, complement was destroyed by heating at 56 °C for 30 minutes, A.F.P. was found to be stable to this treatment. All samples were assayed at three dilutions (1/20, 1/10, and 1/5) in duplicate, and frequently the assay was repeated. Samples from patients with P.H.C.C. required a much higher dilution of their plasma or serum for the A.F.P. level to be measurable by this sensitive R.I.A. method. Serial dilutions of edetic acid (E.D.T.A.), heparin, merthiolate, and sodium azide were incubated in the conditions of a normal assay to investigate the non-specific interference of these reagents with the binding of 115I-A.F.P. by antibody. The specificity of the assay was tested by assessing the immunological cross-reactivity of human serum-albumin, IgG, human chorionic gonadotrophin, transferrin, Creactive protein, a human cord serum (FS-A) and a commercial reference serum (Hoechst Laboratories). The " within " assay variation was estimated by measuring the A.F.P. content of eleven plasma samples twice in the same assay, the " between " assay variation by assaying twelve samples on two different occasions. Recovery of A.F.P. added to a sample was measured by assaying twenty-five plasma samples before and after the addition of 2 or 8 ng. of A.F.P. per ml. of plasma. Student’s t test and correlation coefficient were used for statistical analysis.ll at
Results
253. 8.
of West African
Methods
P. C. GANGULI
University Department of Surgery and Clinical Division of Gastroenterology, Royal Infirmary, Manchester M13 9WL Summary
were
Schroeder, W. A., Huisman, T. H. J., Shelton, R., Shelton, J. B., Kleihauer, E. F., Dozy, A. M., Robberson, B. Proc. natn. Acad. Sci. U.S.A. 1968, 60, 537. Schroeder, W. A., Huisman, T. H. J. 13th int. Congr. Hœmat. 1970, p. 26. Munich. Pembrey, M. E., Weatherall, D. J., Wood, W., Clegg, J. B. Unpublished. Weatherall, D. J., Clegg, J. B., Na-Nakorn, S., Wasi, P. Br. J. Hœmat. 1969, 16, 251. Marti, H. R. Normale und Anormale Menschliche Hämoglobine; p. 81. Berlin, 1963. Chernoff, A. I. Blood, 1953, 8, 413. Schroeder, W. A., Huisman, T. H. J., Shelton, J. R., Wilson, J. B. Analyt. Biochem. 1970, 35, 235. Bromberg, Y. M., Salzberger, M., Abrahamov, A. Blood, 1957, 12, 1122.
16. La Torretta, G., Del Bianco, C. Archs Obstet. Ginec. 1959, 64, 365. 17. Beutler, E. J. Am. med. Ass. 1969, 207, 2284. 18. Perrine, R. P., Brown, M. J., Clegg, J. B., Weatherall, D. J., May, A. Lancet, 1972, ii, 1163.
Radioimmunoassay Method 80% of the 125I-A.F.P. was bound in antibody excess and about 50 % was bound by an antiserum dilution of 1/120,000. A typical standard inhibition curve is shown in fig. 1. The linear portion of the curve covered the range 100 to 3200 pg. of A.F.P. The minimum amount measured in this assay was 100 pg. A.F.P. per ml. of incubate; the maximum concentration of plasma incorporated into the assay without non-specific interference was 80%, thus the sensitivity of this assay was 125 pg. of A.F.P. per ml. of plasma. The precision of the assay at this level was ±7.5% (coefficient of variation). The within " assay and " between " assay variations were "
1356
14
Fig. 3—A.F.P. level, in controls, patients with secondary liver tumours, extrahepatic neoplasms, and P.H.C.C. Arithmetic means±S.D. (log scale) are shown.
Fig. 2-Correlation of A.F.P. with age in years grouped in decades and
sex.
7-5% and 12-2%, respectively. The recovery of added 95-114%. Human serum-albumin, immunoglobulin G, human chorionic gonadotrophin, C-reactive protein, and
A.F.P. was
transferrin did not cross-react with A.F.P. at the concentrations present in the normal serum. The sample dilution curves for the cord serum A, serum from a patient with P.H.C.C., and the commercial reference serum (Hoechst) were linear and parallel to the standard inhibition curve, thus there was no immunological dissimilarity between our standard and the A.F.P. present in these samples. Merthiolate, sodium azide, and heparin did not interfere in this assay, but molarities of E.D.T.A. greater than 2-5 mM altered the binding of 1251-A.F.P. by antibody. There was no significant difference between the A.F.P. level of heparinised plasma and serum prepared from the same sample of blood. Plasma-A.F.P. Level in Patients The controls had an A.F.P. level of 2-3 ±1’2 ng. per ml. of plasma (mean ±S.D.). The upper limit of the " normal range " (mean+3 s.D.) was 5-9 ng. per ml. A significant correlation (r==0’451, P<0.001) was found between age and plasma-A.F.P. level in these 105 controls (fig. 2). The mean values in males (2-4 ±1-3 ng. per ml.) and in females (2.2±1.1ng. per ml.) did not differ significantly. In patients with liver metastases and with extrahepatic malignancies, the A.F.P. levels were 36 ±3.2 ng. per ml. and 2°0 ±2.0 ng. per ml., respectively. There was no significant difference
between the values for these two groups and the control group (fig. 3). Among the 22 patients with P.H.C.C., plasma-A.F.P. was within the normal range in 2-one European and one West African. A.F.P. was slightly raised (less than 25 ng. per ml.) in 3 patients, all from West Africa, but in the remaining 17 patients the A.F.P. level was 400 or more times the upper limit of normal. Discussion
The sensitivity of the methods used for the detection of A.F.P. are about 5 µg. per ml. for immunodiffusion in agar gel,12100 ng. per ml. for immunoradiography or counterimmunoelectrophoresis, 1-11 30 ng. per ml. for passive hæmagglutination,12and 2-5 ng. per ml. for R.I.A.7,8 This assay detects 125 pg. A.F.P. per ml. of plasma with a precision of 7-5% (coefficient of variation). A.F.P. did not cross-react with albumin, the globulins, or glycoproteins normally present in plasma. As in other polypeptide hormone R.I.A.S, E.D.T.A. affected this assay, but merthiolate, heparin, and sodium azide did not affect the primary binding of antigen and antibody.13 The precision of this A.F.P. assay is similar to R.I.A. for polypeptide hormones.14 Plasma-A.F.P. levels in our controls were lower than those reported in other studies, 7,8 perhaps because our patients were screened to exclude those who had clinical or biochemical evidence of chronic liver disease (serum-A.F.P. levels being raised in these disorders 15). There was no difference between the plasma-A.F.P. level of males or females, but there was a rise of A.F.P. levels with age both for males and females; we cannot explain this previously unreported finding. Alpert et al.detected A.F.P. by immunodiffusion in 50 % of 62 patients with P.H.C.C. However, when these same samples were assayed by the more sensitive
1357
PEPTIC ULCERATION IN CHRONIC RENAL FAILURE
technique of counterimmunoelectrophoresis, the test was positive in 72% of the cases. Since this R.I.A. can measure accurately the plasma-A.F.P. level of healthy was hoped that there would be a further increase in the diagnostic value of this test. However, 9% of patients with P.H.C.C. had an A.F.P. level within the normal range, a similar finding to that of Ruoslahti et al.," thus the diagnosis of P.H.C.C. cannot be refuted by a normal A.F.P. level. It is possible that in such patients high values may appear with the progress of the disease, although there is a lot of evidence against this obtained with previous insensitive immunological A.F.P. assays.l3-16 Other workers have failed to show any correlation between serum-A.F.P. level and the degree of differentiation of the tumour or the clinical evolution of the disease.13,16,18It is probable that the
W. K. STEWART A. M. M. SHEPHERD K. G. WORMSLEY Renal and Gastroenterological Units, Maryfield Hospital, Dundee DD4 7TL
adults, it
produced by some P.H.c.c.s is immunologically dissimilar to human fetal A.F.P., which was the starting material for the reagents used in this assay. It may be fruitful to develop an R.I.A. starting with A.F.P. extracted from the serum or tumour of patients with P.H.C.C. The early workers who were unable to detect A.F.P. in normal serum held the view that a raised A.F.P. level was diagnostic of P.H.c.c. Since more sensitive methods have been developed, raised A.F.P. levels have been encountered with teratoblastomas of the gonads, 3-6, 17 ataxia-telangiectasia,infectious hepatitis,15 lsecondary metastatic liver tumours,15 and, rarely, certain carcinomas of the stomach.19 In our series, none of the patients with liver metastases or extrahepatic neoplasms had a raised A.F.P. level; however, further observations on such groups are advisable. It has been suggested that soon after birth there is " repression " of the genes responsible for coding for A.F.P. synthesis during fetal life. 20 The reappearance of A.F.P. in the serum of patients with P.H.c.c. may be due to the " derepression " of these genes by malignant liver cells. 3,4 The fact that small amounts of A.F.P. can be detected in the serum of normal adults 7, by sensitive R.I.A. methods suggests that the repression of A.F.P. synthesis at birth is not complete. It is likely that most P.H.C.C.s develop from the " fetal type " population of liver cells, and their increased activity may be responsible for the raised A.F.P. levels encountered in patients with P.H.c.c. The application of a sensitive radioimmunoassay for A.F.P. will make it possible to investigate the effect of liver damage, chronic liver disease, and primary hepatocellular carcinoma on this fetal protein. A.F.P.
We thank Dr E. Ruoslahti, Helsinki, for donating the highly A.F.P. and sheep anti-human A.F.P. serum; Dr R. Harris, Manchester, for his encouragement and help; Dr J. Uriel, Paris, and Dr J. A. Smith, Nigeria, for sending us serum samples from patients with proven P.H.C.C.; Dr W. M. Hunter, Edinburgh, for the human chorionic gonadotrophin, and Dr H. McFarlane, Manchester, for the human serum albumin, IgG, transferrin, and C-reactive protein. Prof. I. E. Gillespie and Mr J. B. Elder, Prof. H. T. Howat, and Mr H. B. Torrance, Manchester, kindly allowed us to study their patients and helped with the manuscript. We thank our many colleagues for helping us to collect the clinical material for these studies. The Department of Medical Illustration prepared the figures. We thank the Medical Research Council and the Institut National de la Santé et de la Recherche Medicale, France, for supporting J. A. P. C. by means of an Anglo-French clinical research scholarship. Requests for reprints should be addressed to P. C. G.
purified
patients with chronic renal failure maintenance hæmodialysis, very high overnight and basal gastric-acid secretion was associated with an extremely high frequency (53%) of In
Sum ary on
duodenal ulceration.
Since the duodenal ulcer may
clinically manifest, spontaneous gastric-acid secretion should be measured in all patients with chronic renal insufficiency, so that treatment can be given if gastric hypersecretion is present. not
be
Introduction
Dekkers et al.1,2reported an extraordinarily high nocturnal secretion of acid in patients with chronic renal failure. We have investigated gastric secretion in patients undergoing maintenance hxmodialysis, in whom the frequency of duodenal ulceration was
extremely high. Methods 15 patients with end-stage chronic renal failure (7 with chronic glomerulonephritis, 3 with analgesic nephropathy, 2 with chronic pyelonephritis, 1 with polycystic disease, 1 with hereditary chronic nephritis, and 1 with undefined chronic renal disease) receiving chronic maintenance hæmodialysis were studied. Routine disposable-coil-based hasmodialysis had been in progress for three to sixty months. Duodenal ulceration was confirmed radiologically or at
laparotomy.
Bergstrand, C. G., Czar, B. Scand. J. clin. Lab. Invest. 1956, 8, 174. Tatarinov, J. Vopr. Med. Khim. 1964, 10, 90. Abelev, G. I., Assecritova, I. V., Kraevsky, N. A., Perova, S. D., Perevodchikova, N. I. Int. J. Cancer, 1967, 2, 551. 4. Uriel, J., de Nechaud, B., Stanislawski-Birencwajg, M., Masseyef, R., Leblanc, L., Quenum, C., Loisillier, F., Grabar, P. Presse méd. 1968, 76, 1415. 5. Alpert, M. E., Uriel, J., de Nechaud, B. New Engl. J. Med. 1968, 278, 984. 6. Alpert, E., Hershberg, R., Schur, P. H., Isselbacher, K. J. Gastroenterology, 1971, 61, 137. 7. Ruoslahti, E., Seppälä, M. Int. J. Cancer, 1971, 8, 374. 8. Waldmann, T. A., McIntire, K. R. Lancet, 1972, ii, 1112. 9. Hunter, W. M., Greenwood, F. C. Nature, 1962, 194, 495. 10. Hunter, W. M., Ganguli, P. C. in Radioimmunoassay Workshop (edited by K. E. Kirkham and W. M. Hunter); p. 243. Edinburgh, 1. 2. 3.
1971. 11.
Snedecor, G. W., Cochran, W.
G. Statistical Methods. Ames, Iowa,
1967. 12.
Lehmann,
F.
G., Lehmann, D., Martini, G. A. Digestion, 1972, 6,
249. 13.
Abelev, G. I., Tsvetkov, V. S., Biryulina, T. I., El’Gort, D. A., Olovnikov, A. M., Gusev, A. I., Yazova, A. K., Perova, S. D., Rubtsov, I. V., Shaborina, S. V., Kantorovich, B. A., Tur, V. M., Khazanov, A. I., Levina, D. M. Byull. Eksp. Biol. Med. 1971, 71, 75.
14. Ganguli, P. C., Hunter, W. M. J. Physiol., Lond. 1972, 202, 499. 15. Ruoslahti, E., Seppälä, M., Vuopio, P., Saksela, E., Peltokallio, P. J. natn. Cancer Inst. 1972, 49, 623. 16. McIntire, K. R., Vogel, C. L., Princler, G. L., Patel, I. L. Cancer Res. 1972, 32, 1941. 17. Masopust, J., Kithier, K., Koutecky, J., Kotal, L. Int. J. Cancer, 1968, 3, 364. 18. Purves, L. R., Bersohn, I., Geddes, E. W. Cancer, 1970, 25, 1261. 19. Boureille, J., Metayer, P., Sauger, F., Matray, F., Fondimare, A. Presse méd. 1970, 78, 1277. 20. Gitlin, D., Boesman, M. J. clin. Invest. 1966, 45, 1826.
neoplasms. 5 of the patients with
RADIOIMMUNOASSAY OF ALPHAFETOPROTEIN IN HUMAN PLASMA
J.
A. P. CHAYVIALLE
the other 17
A
highly sensitive radioimmunoassay capable of measuring &agr;1-fetoprotein been has developed. A.F.P. was detected in the (A.F.P.) all 105 controls (2·3±1·2 ng. per ml.). of the plasma There was no significant difference between the A.P.P. level of controls and those with histologically proven secondary liver tumours (13 patients) or extrahepatic neoplasms (23 patients). In 17 of the 22 patients (77%) with primary hepatocellular carcinoma, the A.F.P. level was very elevated (2·5 µg. per ml. to 2·6 mg. per ml.), in another 3 patients (14%) it was moderately high (10-25 ng. per ml.), while in 2 of the 22 patients (9%) it
was
within the normal range. Introduction
THE
serum
of the human fetus contains
an
OC1-
globulin called oc-fetoprotein (A.F.P.). Using insensitive immunological methods A.F.P. was detected in 40-80% of the sera of patients with primary hepatocellular carcinoma (P.H.C.c.) but not in controls .2-5 The percentage of positive results was higher when more sensitive techniques were used for the estimation of A.F.P.6A.F.P. has been measured by radioimmunoassay. 7 ,We report here plasma-A.F.P. levels in controls and in patients with P.H.C.C. and liver metastases or extrahepatic malignancies arising from the gut, lungs, thyroid, prostate, or pituitary gland, using a highly sensitive radioimmunoassay (R.I.A.) method. Patients and Methods
Patients We studied 163 patients. There were equal numbers of males and females. 105 patients were hospital controls with no clinical or biochemical evidence of liver disease. The other 58 patients had histologically proven tumours22 were P.H.C.C., 13 liver metastases, and 23 extrahepatic
Pembrey, M. E., Weatherall, D. J., McWade, P. J. clin. Path. 1972, 25, 738. 6. Kleihauer, E., Braun, H., Betke, K. Klin. Wschr. 1957, 35, 637. 7. Weatherall, D. J., Gilles, H. M., Clegg, J. B., Blankson, J. A., Mustafa, D., Boi-Doku, F. S. Ann. trop. Med. Parasit. 1971, 65, 5.
9. 10. 11.
12. 13. 14. 15.
P.H.C.C. were
Europeans,
origin.
The highly purified A.F.P. used as standard and for radioiodination and the sheep anti-human A.F.P. serum were both donated by Dr E. Ruoslahti. A.F.P. was iodinated with iodine-125 (Radiochemicals Centre, Amersham) using the chloramine-T method of Hunter and Greenwood.9 The sheep anti-human A.F.P. serum was titrated by incubating it in serial dilutions (1/5000 to 1/2,560,000) -with 50 pg. 225I-A.F.P. in 005M phosphate buffer, pH 7-5, with 1% horse serum, at 4°C for 6 days. Antibody-bound 12sI-A.F.P. was separated from free 125I-A.F.P. by a double-antibody method’IO using 1/20,000 non-immune sheep serum and a rabbit anti-goat gamma-globulin serum (As 322, Wellcome Reagents) at a final dilution of 1/200. 24 hours later, after adding cold diluent, the tubes were centrifuged at 2000 g at 4°C for 60 minutes. The supernatant was discarded and the radioactivity of the precipitate counted in an automatic gamma spectrometer (Wallac). Standard inhibition curves were obtained by incubating serial amounts of standard A.F.P. (50-3200 pg.) with antiserum at a final dilution of 1/120,000 at 4°C for 2 days. 50 pg. 125I-A.F.P. was then added to each tube and they were incubated for a further 48 hours. Antibody-bound and free 115I-A.F.P. were
separated. Plasma or serum was obtained from patients and stored -30°C. Before assay, complement was destroyed by heating at 56 °C for 30 minutes, A.F.P. was found to be stable to this treatment. All samples were assayed at three dilutions (1/20, 1/10, and 1/5) in duplicate, and frequently the assay was repeated. Samples from patients with P.H.C.C. required a much higher dilution of their plasma or serum for the A.F.P. level to be measurable by this sensitive R.I.A. method. Serial dilutions of edetic acid (E.D.T.A.), heparin, merthiolate, and sodium azide were incubated in the conditions of a normal assay to investigate the non-specific interference of these reagents with the binding of 115I-A.F.P. by antibody. The specificity of the assay was tested by assessing the immunological cross-reactivity of human serum-albumin, IgG, human chorionic gonadotrophin, transferrin, Creactive protein, a human cord serum (FS-A) and a commercial reference serum (Hoechst Laboratories). The " within " assay variation was estimated by measuring the A.F.P. content of eleven plasma samples twice in the same assay, the " between " assay variation by assaying twelve samples on two different occasions. Recovery of A.F.P. added to a sample was measured by assaying twenty-five plasma samples before and after the addition of 2 or 8 ng. of A.F.P. per ml. of plasma. Student’s t test and correlation coefficient were used for statistical analysis.ll at
Results
253. 8.
of West African
Methods
P. C. GANGULI
University Department of Surgery and Clinical Division of Gastroenterology, Royal Infirmary, Manchester M13 9WL Summary
were
Schroeder, W. A., Huisman, T. H. J., Shelton, R., Shelton, J. B., Kleihauer, E. F., Dozy, A. M., Robberson, B. Proc. natn. Acad. Sci. U.S.A. 1968, 60, 537. Schroeder, W. A., Huisman, T. H. J. 13th int. Congr. Hœmat. 1970, p. 26. Munich. Pembrey, M. E., Weatherall, D. J., Wood, W., Clegg, J. B. Unpublished. Weatherall, D. J., Clegg, J. B., Na-Nakorn, S., Wasi, P. Br. J. Hœmat. 1969, 16, 251. Marti, H. R. Normale und Anormale Menschliche Hämoglobine; p. 81. Berlin, 1963. Chernoff, A. I. Blood, 1953, 8, 413. Schroeder, W. A., Huisman, T. H. J., Shelton, J. R., Wilson, J. B. Analyt. Biochem. 1970, 35, 235. Bromberg, Y. M., Salzberger, M., Abrahamov, A. Blood, 1957, 12, 1122.
16. La Torretta, G., Del Bianco, C. Archs Obstet. Ginec. 1959, 64, 365. 17. Beutler, E. J. Am. med. Ass. 1969, 207, 2284. 18. Perrine, R. P., Brown, M. J., Clegg, J. B., Weatherall, D. J., May, A. Lancet, 1972, ii, 1163.
Radioimmunoassay Method 80% of the 125I-A.F.P. was bound in antibody excess and about 50 % was bound by an antiserum dilution of 1/120,000. A typical standard inhibition curve is shown in fig. 1. The linear portion of the curve covered the range 100 to 3200 pg. of A.F.P. The minimum amount measured in this assay was 100 pg. A.F.P. per ml. of incubate; the maximum concentration of plasma incorporated into the assay without non-specific interference was 80%, thus the sensitivity of this assay was 125 pg. of A.F.P. per ml. of plasma. The precision of the assay at this level was ±7.5% (coefficient of variation). The within " assay and " between " assay variations were "
1356
14
Fig. 3—A.F.P. level, in controls, patients with secondary liver tumours, extrahepatic neoplasms, and P.H.C.C. Arithmetic means±S.D. (log scale) are shown.
Fig. 2-Correlation of A.F.P. with age in years grouped in decades and
sex.
7-5% and 12-2%, respectively. The recovery of added 95-114%. Human serum-albumin, immunoglobulin G, human chorionic gonadotrophin, C-reactive protein, and
A.F.P. was
transferrin did not cross-react with A.F.P. at the concentrations present in the normal serum. The sample dilution curves for the cord serum A, serum from a patient with P.H.C.C., and the commercial reference serum (Hoechst) were linear and parallel to the standard inhibition curve, thus there was no immunological dissimilarity between our standard and the A.F.P. present in these samples. Merthiolate, sodium azide, and heparin did not interfere in this assay, but molarities of E.D.T.A. greater than 2-5 mM altered the binding of 1251-A.F.P. by antibody. There was no significant difference between the A.F.P. level of heparinised plasma and serum prepared from the same sample of blood. Plasma-A.F.P. Level in Patients The controls had an A.F.P. level of 2-3 ±1’2 ng. per ml. of plasma (mean ±S.D.). The upper limit of the " normal range " (mean+3 s.D.) was 5-9 ng. per ml. A significant correlation (r==0’451, P<0.001) was found between age and plasma-A.F.P. level in these 105 controls (fig. 2). The mean values in males (2-4 ±1-3 ng. per ml.) and in females (2.2±1.1ng. per ml.) did not differ significantly. In patients with liver metastases and with extrahepatic malignancies, the A.F.P. levels were 36 ±3.2 ng. per ml. and 2°0 ±2.0 ng. per ml., respectively. There was no significant difference
between the values for these two groups and the control group (fig. 3). Among the 22 patients with P.H.C.C., plasma-A.F.P. was within the normal range in 2-one European and one West African. A.F.P. was slightly raised (less than 25 ng. per ml.) in 3 patients, all from West Africa, but in the remaining 17 patients the A.F.P. level was 400 or more times the upper limit of normal. Discussion
The sensitivity of the methods used for the detection of A.F.P. are about 5 µg. per ml. for immunodiffusion in agar gel,12100 ng. per ml. for immunoradiography or counterimmunoelectrophoresis, 1-11 30 ng. per ml. for passive hæmagglutination,12and 2-5 ng. per ml. for R.I.A.7,8 This assay detects 125 pg. A.F.P. per ml. of plasma with a precision of 7-5% (coefficient of variation). A.F.P. did not cross-react with albumin, the globulins, or glycoproteins normally present in plasma. As in other polypeptide hormone R.I.A.S, E.D.T.A. affected this assay, but merthiolate, heparin, and sodium azide did not affect the primary binding of antigen and antibody.13 The precision of this A.F.P. assay is similar to R.I.A. for polypeptide hormones.14 Plasma-A.F.P. levels in our controls were lower than those reported in other studies, 7,8 perhaps because our patients were screened to exclude those who had clinical or biochemical evidence of chronic liver disease (serum-A.F.P. levels being raised in these disorders 15). There was no difference between the plasma-A.F.P. level of males or females, but there was a rise of A.F.P. levels with age both for males and females; we cannot explain this previously unreported finding. Alpert et al.detected A.F.P. by immunodiffusion in 50 % of 62 patients with P.H.C.C. However, when these same samples were assayed by the more sensitive
1357
PEPTIC ULCERATION IN CHRONIC RENAL FAILURE
technique of counterimmunoelectrophoresis, the test was positive in 72% of the cases. Since this R.I.A. can measure accurately the plasma-A.F.P. level of healthy was hoped that there would be a further increase in the diagnostic value of this test. However, 9% of patients with P.H.C.C. had an A.F.P. level within the normal range, a similar finding to that of Ruoslahti et al.," thus the diagnosis of P.H.C.C. cannot be refuted by a normal A.F.P. level. It is possible that in such patients high values may appear with the progress of the disease, although there is a lot of evidence against this obtained with previous insensitive immunological A.F.P. assays.l3-16 Other workers have failed to show any correlation between serum-A.F.P. level and the degree of differentiation of the tumour or the clinical evolution of the disease.13,16,18It is probable that the
W. K. STEWART A. M. M. SHEPHERD K. G. WORMSLEY Renal and Gastroenterological Units, Maryfield Hospital, Dundee DD4 7TL
adults, it
produced by some P.H.c.c.s is immunologically dissimilar to human fetal A.F.P., which was the starting material for the reagents used in this assay. It may be fruitful to develop an R.I.A. starting with A.F.P. extracted from the serum or tumour of patients with P.H.C.C. The early workers who were unable to detect A.F.P. in normal serum held the view that a raised A.F.P. level was diagnostic of P.H.c.c. Since more sensitive methods have been developed, raised A.F.P. levels have been encountered with teratoblastomas of the gonads, 3-6, 17 ataxia-telangiectasia,infectious hepatitis,15 lsecondary metastatic liver tumours,15 and, rarely, certain carcinomas of the stomach.19 In our series, none of the patients with liver metastases or extrahepatic neoplasms had a raised A.F.P. level; however, further observations on such groups are advisable. It has been suggested that soon after birth there is " repression " of the genes responsible for coding for A.F.P. synthesis during fetal life. 20 The reappearance of A.F.P. in the serum of patients with P.H.c.c. may be due to the " derepression " of these genes by malignant liver cells. 3,4 The fact that small amounts of A.F.P. can be detected in the serum of normal adults 7, by sensitive R.I.A. methods suggests that the repression of A.F.P. synthesis at birth is not complete. It is likely that most P.H.C.C.s develop from the " fetal type " population of liver cells, and their increased activity may be responsible for the raised A.F.P. levels encountered in patients with P.H.c.c. The application of a sensitive radioimmunoassay for A.F.P. will make it possible to investigate the effect of liver damage, chronic liver disease, and primary hepatocellular carcinoma on this fetal protein. A.F.P.
We thank Dr E. Ruoslahti, Helsinki, for donating the highly A.F.P. and sheep anti-human A.F.P. serum; Dr R. Harris, Manchester, for his encouragement and help; Dr J. Uriel, Paris, and Dr J. A. Smith, Nigeria, for sending us serum samples from patients with proven P.H.C.C.; Dr W. M. Hunter, Edinburgh, for the human chorionic gonadotrophin, and Dr H. McFarlane, Manchester, for the human serum albumin, IgG, transferrin, and C-reactive protein. Prof. I. E. Gillespie and Mr J. B. Elder, Prof. H. T. Howat, and Mr H. B. Torrance, Manchester, kindly allowed us to study their patients and helped with the manuscript. We thank our many colleagues for helping us to collect the clinical material for these studies. The Department of Medical Illustration prepared the figures. We thank the Medical Research Council and the Institut National de la Santé et de la Recherche Medicale, France, for supporting J. A. P. C. by means of an Anglo-French clinical research scholarship. Requests for reprints should be addressed to P. C. G.
purified
patients with chronic renal failure maintenance hæmodialysis, very high overnight and basal gastric-acid secretion was associated with an extremely high frequency (53%) of In
Sum ary on
duodenal ulceration.
Since the duodenal ulcer may
clinically manifest, spontaneous gastric-acid secretion should be measured in all patients with chronic renal insufficiency, so that treatment can be given if gastric hypersecretion is present. not
be
Introduction
Dekkers et al.1,2reported an extraordinarily high nocturnal secretion of acid in patients with chronic renal failure. We have investigated gastric secretion in patients undergoing maintenance hxmodialysis, in whom the frequency of duodenal ulceration was
extremely high. Methods 15 patients with end-stage chronic renal failure (7 with chronic glomerulonephritis, 3 with analgesic nephropathy, 2 with chronic pyelonephritis, 1 with polycystic disease, 1 with hereditary chronic nephritis, and 1 with undefined chronic renal disease) receiving chronic maintenance hæmodialysis were studied. Routine disposable-coil-based hasmodialysis had been in progress for three to sixty months. Duodenal ulceration was confirmed radiologically or at
laparotomy.
Bergstrand, C. G., Czar, B. Scand. J. clin. Lab. Invest. 1956, 8, 174. Tatarinov, J. Vopr. Med. Khim. 1964, 10, 90. Abelev, G. I., Assecritova, I. V., Kraevsky, N. A., Perova, S. D., Perevodchikova, N. I. Int. J. Cancer, 1967, 2, 551. 4. Uriel, J., de Nechaud, B., Stanislawski-Birencwajg, M., Masseyef, R., Leblanc, L., Quenum, C., Loisillier, F., Grabar, P. Presse méd. 1968, 76, 1415. 5. Alpert, M. E., Uriel, J., de Nechaud, B. New Engl. J. Med. 1968, 278, 984. 6. Alpert, E., Hershberg, R., Schur, P. H., Isselbacher, K. J. Gastroenterology, 1971, 61, 137. 7. Ruoslahti, E., Seppälä, M. Int. J. Cancer, 1971, 8, 374. 8. Waldmann, T. A., McIntire, K. R. Lancet, 1972, ii, 1112. 9. Hunter, W. M., Greenwood, F. C. Nature, 1962, 194, 495. 10. Hunter, W. M., Ganguli, P. C. in Radioimmunoassay Workshop (edited by K. E. Kirkham and W. M. Hunter); p. 243. Edinburgh, 1. 2. 3.
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G. Statistical Methods. Ames, Iowa,
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Abelev, G. I., Tsvetkov, V. S., Biryulina, T. I., El’Gort, D. A., Olovnikov, A. M., Gusev, A. I., Yazova, A. K., Perova, S. D., Rubtsov, I. V., Shaborina, S. V., Kantorovich, B. A., Tur, V. M., Khazanov, A. I., Levina, D. M. Byull. Eksp. Biol. Med. 1971, 71, 75.
14. Ganguli, P. C., Hunter, W. M. J. Physiol., Lond. 1972, 202, 499. 15. Ruoslahti, E., Seppälä, M., Vuopio, P., Saksela, E., Peltokallio, P. J. natn. Cancer Inst. 1972, 49, 623. 16. McIntire, K. R., Vogel, C. L., Princler, G. L., Patel, I. L. Cancer Res. 1972, 32, 1941. 17. Masopust, J., Kithier, K., Koutecky, J., Kotal, L. Int. J. Cancer, 1968, 3, 364. 18. Purves, L. R., Bersohn, I., Geddes, E. W. Cancer, 1970, 25, 1261. 19. Boureille, J., Metayer, P., Sauger, F., Matray, F., Fondimare, A. Presse méd. 1970, 78, 1277. 20. Gitlin, D., Boesman, M. J. clin. Invest. 1966, 45, 1826.