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BIOLOGICAL ACTIVITY OF HEPATITIS-BANTIGEN SOLUBLE IMMUNE COMPLEXES
968 Whites. The medical services in the rural areas are pracnon-existent: rural Africans in many less-developed African countries have easier access to medical care than those in South Africa. Very few Africans are being trained as doctors. Coloureds and Indians who are " privileged " to attend White universities (if they obtain consent from the Minister of Education) receive inferior training because they are confined to Black hospitals, where facilities are poor. Last year a group of about twenty third-year White medical students at the University of Witwatersrand signed a petition requesting that in their clinical years they should be treated as Black students. The heads of the clinical departments were at great pains to impress on the students that they would as a result be handicapped in their training -which amounts to an admission that the Blacks receive inferior training. A stamp of approval is given to the status quo in South Africa every time a medical authority visits South Africa or South African Whites are allowed to attend international conferences. It is extremely difficult for Blacks to protest in South Africa for fear of reprisals; silence on the part of the Blacks is no indication of their content with the present The onus, therefore, is on the international system. organisations-particularly British and American organisations-to boycott South Africa until racial discrimination has been eliminated from medicine there.
tically
A BLACK SOUTH AFRICAN.
BIOLOGICAL ACTIVITY OF HEPATITIS-BANTIGEN SOLUBLE IMMUNE COMPLEXES
SIR,-It is suspected that hepatitis-B infection
may be
complicated in certain cases by the development of an immune-complex syndrome.1-4 This letter demonstrates that soluble HBAg-antibody complexes formed in vitro and tested intradermally in guineapigs’ skin have considerable biological activity. Antigen-excess and equivalence-zone HBAg-antibody complexes were formed by addition of appropriate volumes of hyperimmune anti-HBAg to HBAg-positive human serum. The mixtures were incubated for 1 hour at 37 °C and subsequently for 48 hours at 4°C. Precipitates and soluble HBAg-antibody complexes were separated from free HBAg by overlaying the mixtures over a discontinuous sucrose gradient (15-50%) in phosphate buffer pH 7-4. After ultracentrifugation for 1 hour at 26,384 g at 4°C, four drop fractions were collected from the bottom and tested for antigen and antibody activity in immunodiffusion plates. Fractions containing complexes were pooled, diluted in phosphate-buffered saline, and re-centrifuged for 16 hours at 26,384 g. Pellets were taken up in 0.5 ml. of saline and the protein content measured by the method of Lowry et al.I Complexes formed at equivalence and in antigen excess were injected intradermally at multiple sites (400 and 800 g. protein per site) on the backs of male albino guineapigs prepared with 1 % Evans blue dye. The reactions were observed and compared with controls from 30 minutes to 3 hours by measuring the diameter of blueing under transillumination. HBAg-antibody complexes in antigen excess produced immediate increased vascular permeability and fourfold to fivefold larger reactions than control injections (16-22 mm. v. 2-4 No significant difference between equivalence-zone mm.). precipitates and controls was recognisable even after 3 hours. These preliminary findings demonstrate the capacity of soluble
HBAg-HBAb complexes to cause tissue injury, and provide further basis for investigation of the role of such complexes in the production of intrahepatic and extrahepatic damage in man. Department of Medicine, Rutgers Medical School, New Brunswick, New Jersey, U.S.A.
EFFECT OF FOOD ON ZINC ABSORPTION
SIR,-Dr Reinhold (Feb. 10, p. 283) has shown that phytate increases zinc losses in fasces, and both Professor Prasad (June 30, p. 1520) and Dr Reinhold (July 7, p. 50), in reviewing the problem of zinc deficiency due to phytaterich food and associated with retarded growth in the Middle East, mentioned that very low amounts of zinc (30 mg. daily) are sufficient to compensate for such deficiencies if the food is poor in phytates. Much higher doses of zinc (600 mg. ZnS04.7H2O, equivalent to 150 mg. of zinc++) have been given daily for several weeks to patients with indolent surgical wounds or chronic leg ulcers. Some beneficial effect on the healing-rate was reported 1-4; the results of other studies were negative.5.6 Serum-zinc concentrations rose significantly,2-4 but some data showed rather large discrepancies. 4,5,7 These patients were on " standard, Western ", diet, which is supposed to be poor in phytate. However, zinc sulphate was administered orally, in three divided doses of 220 mg., and was taken either with the meals,4 or after the meals,l, 2.5-7 to minimise gastric irritation. This mode of administration might have influenced the absorption of zinc, as suggested by the following data.
Healthy young volunteers were allocated to three groups of six subjects who were given two zinc salts, either in the fasting state, or with a breakfast (milk, coffee, bread, butter, jelly, cheese), or 45 minutes after the same type of breakfast. A placebo (lactose) was given on the first day, one zinc preparation on the 2nd day, and the other zinc preparation at least a week later. Zinc was administered either as ZnS04.7H20, 220 mg. (50 mg. of zinc++) or as the zinc salt of N-acetylhydroxyproline, 320 mg. (50 mg. of zinc++), supplied by Merrell International Research Centre, Strasbourg, France. The placebo and the drugs were prepared in small identical gelatin capsules by the hospital pharmacist. They were taken with a glass of water by the subjects who were fasting and those who took the drug after the breakfast. Each experiment was started at 8 A.M., the subjects having fasted for more than 10 hours. Blood-samples were drawn in zinc-free plastic material, and serum-zinc concentrations were determined by atomic-absorption spectrophotometry.e The serum-zinc concentrations rose to a higher level in fasting subjects than in those who took the drugs after breakfast, whereas the values measured when the drugs were taken with the breakfast were unchanged. Mean serum-zinc concentrations (S.E.M.) 3 hours after administration of zinc sulphate were 270:f:: 18 g. per 100 ml. in the fasting group, 146 :f::28 Ilg. per 100 ml. in the after breakfast " group, and 73±4 tig. per 100 ml. in the " with breakfast" group. The differences between the first and the second group were significant (mean difference 124 g. per 100 ml.; S.E.M. of pooled observations 33; 10 degrees of freedom; p < 001), as were the differences between the second and the third group (73 ±28 gg. per 100 ml.; 10 degrees of freedom; p < 0-05). The differences between groups for the zinc salt of N-acetylhydroxyproline were also significant. On the other hand, the values obtained with the two zinc salts in the same conditions did not differ significantly from each other. In the fasting group, one subject vomited after zinc sulphate and "
three had definite
Gocke, D. J., Hsu, K., Morgan, C., Bombardieri, S., Lockshin, M., Christian, C. Lancet, 1970, ii, 1149. 2. Alpert, E., Isselbacher, K. J., Schur, P. H. New Engl. J. Med. 1971, 285, 185. 3. Onion, D. K., Crumpacker, C. S., Gilliland, B. C. Ann. intern. Med. 1971, 75, 29. 4. Combes, B., Stastny, P., Shorey, J., Eigenbrodt, E. H., Barrera, A., Hull, A. R., Carter, W. W. Lancet, 1971, ii, 234. 5. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. J. biol. Chem. 1951, 193, 265.
V. J. McAULIFFE Z. F. KACHANI D. J. GOCKE.
nausea
after the other zinc salt; such
severe
1.
Pories, W. J., Henzel, J. H., Rob, C. G., Strain, W. H. Lancet, 1967, i, 121. 2. Husain, S. L. ibid. 1969, i, 1069. 3. Serjeant, G. R., Galloway, R. E., Gueri, M. C. ibid. 1970, ii, 891. 4. Hallböök, T., Lanner, E. ibid. 1972, ii, 780. 5. Myers, B. M., Cherry, G. Am. J. Surg. 1970, 120, 77. 6. Greaves, M. W., Ive, F. A. Br. J. Derm. 1972, 87, 632. 7. Greaves, M. W., Skillen, A. W. Lancet, 1970, ii, 889. 8. Meret, S., Henkin, R. I. Clin. Chem. 1971, 17, 369. 1.
tically
A BLACK SOUTH AFRICAN.
BIOLOGICAL ACTIVITY OF HEPATITIS-BANTIGEN SOLUBLE IMMUNE COMPLEXES
SIR,-It is suspected that hepatitis-B infection
may be
complicated in certain cases by the development of an immune-complex syndrome.1-4 This letter demonstrates that soluble HBAg-antibody complexes formed in vitro and tested intradermally in guineapigs’ skin have considerable biological activity. Antigen-excess and equivalence-zone HBAg-antibody complexes were formed by addition of appropriate volumes of hyperimmune anti-HBAg to HBAg-positive human serum. The mixtures were incubated for 1 hour at 37 °C and subsequently for 48 hours at 4°C. Precipitates and soluble HBAg-antibody complexes were separated from free HBAg by overlaying the mixtures over a discontinuous sucrose gradient (15-50%) in phosphate buffer pH 7-4. After ultracentrifugation for 1 hour at 26,384 g at 4°C, four drop fractions were collected from the bottom and tested for antigen and antibody activity in immunodiffusion plates. Fractions containing complexes were pooled, diluted in phosphate-buffered saline, and re-centrifuged for 16 hours at 26,384 g. Pellets were taken up in 0.5 ml. of saline and the protein content measured by the method of Lowry et al.I Complexes formed at equivalence and in antigen excess were injected intradermally at multiple sites (400 and 800 g. protein per site) on the backs of male albino guineapigs prepared with 1 % Evans blue dye. The reactions were observed and compared with controls from 30 minutes to 3 hours by measuring the diameter of blueing under transillumination. HBAg-antibody complexes in antigen excess produced immediate increased vascular permeability and fourfold to fivefold larger reactions than control injections (16-22 mm. v. 2-4 No significant difference between equivalence-zone mm.). precipitates and controls was recognisable even after 3 hours. These preliminary findings demonstrate the capacity of soluble
HBAg-HBAb complexes to cause tissue injury, and provide further basis for investigation of the role of such complexes in the production of intrahepatic and extrahepatic damage in man. Department of Medicine, Rutgers Medical School, New Brunswick, New Jersey, U.S.A.
EFFECT OF FOOD ON ZINC ABSORPTION
SIR,-Dr Reinhold (Feb. 10, p. 283) has shown that phytate increases zinc losses in fasces, and both Professor Prasad (June 30, p. 1520) and Dr Reinhold (July 7, p. 50), in reviewing the problem of zinc deficiency due to phytaterich food and associated with retarded growth in the Middle East, mentioned that very low amounts of zinc (30 mg. daily) are sufficient to compensate for such deficiencies if the food is poor in phytates. Much higher doses of zinc (600 mg. ZnS04.7H2O, equivalent to 150 mg. of zinc++) have been given daily for several weeks to patients with indolent surgical wounds or chronic leg ulcers. Some beneficial effect on the healing-rate was reported 1-4; the results of other studies were negative.5.6 Serum-zinc concentrations rose significantly,2-4 but some data showed rather large discrepancies. 4,5,7 These patients were on " standard, Western ", diet, which is supposed to be poor in phytate. However, zinc sulphate was administered orally, in three divided doses of 220 mg., and was taken either with the meals,4 or after the meals,l, 2.5-7 to minimise gastric irritation. This mode of administration might have influenced the absorption of zinc, as suggested by the following data.
Healthy young volunteers were allocated to three groups of six subjects who were given two zinc salts, either in the fasting state, or with a breakfast (milk, coffee, bread, butter, jelly, cheese), or 45 minutes after the same type of breakfast. A placebo (lactose) was given on the first day, one zinc preparation on the 2nd day, and the other zinc preparation at least a week later. Zinc was administered either as ZnS04.7H20, 220 mg. (50 mg. of zinc++) or as the zinc salt of N-acetylhydroxyproline, 320 mg. (50 mg. of zinc++), supplied by Merrell International Research Centre, Strasbourg, France. The placebo and the drugs were prepared in small identical gelatin capsules by the hospital pharmacist. They were taken with a glass of water by the subjects who were fasting and those who took the drug after the breakfast. Each experiment was started at 8 A.M., the subjects having fasted for more than 10 hours. Blood-samples were drawn in zinc-free plastic material, and serum-zinc concentrations were determined by atomic-absorption spectrophotometry.e The serum-zinc concentrations rose to a higher level in fasting subjects than in those who took the drugs after breakfast, whereas the values measured when the drugs were taken with the breakfast were unchanged. Mean serum-zinc concentrations (S.E.M.) 3 hours after administration of zinc sulphate were 270:f:: 18 g. per 100 ml. in the fasting group, 146 :f::28 Ilg. per 100 ml. in the after breakfast " group, and 73±4 tig. per 100 ml. in the " with breakfast" group. The differences between the first and the second group were significant (mean difference 124 g. per 100 ml.; S.E.M. of pooled observations 33; 10 degrees of freedom; p < 001), as were the differences between the second and the third group (73 ±28 gg. per 100 ml.; 10 degrees of freedom; p < 0-05). The differences between groups for the zinc salt of N-acetylhydroxyproline were also significant. On the other hand, the values obtained with the two zinc salts in the same conditions did not differ significantly from each other. In the fasting group, one subject vomited after zinc sulphate and "
three had definite
Gocke, D. J., Hsu, K., Morgan, C., Bombardieri, S., Lockshin, M., Christian, C. Lancet, 1970, ii, 1149. 2. Alpert, E., Isselbacher, K. J., Schur, P. H. New Engl. J. Med. 1971, 285, 185. 3. Onion, D. K., Crumpacker, C. S., Gilliland, B. C. Ann. intern. Med. 1971, 75, 29. 4. Combes, B., Stastny, P., Shorey, J., Eigenbrodt, E. H., Barrera, A., Hull, A. R., Carter, W. W. Lancet, 1971, ii, 234. 5. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. J. biol. Chem. 1951, 193, 265.
V. J. McAULIFFE Z. F. KACHANI D. J. GOCKE.
nausea
after the other zinc salt; such
severe
1.
Pories, W. J., Henzel, J. H., Rob, C. G., Strain, W. H. Lancet, 1967, i, 121. 2. Husain, S. L. ibid. 1969, i, 1069. 3. Serjeant, G. R., Galloway, R. E., Gueri, M. C. ibid. 1970, ii, 891. 4. Hallböök, T., Lanner, E. ibid. 1972, ii, 780. 5. Myers, B. M., Cherry, G. Am. J. Surg. 1970, 120, 77. 6. Greaves, M. W., Ive, F. A. Br. J. Derm. 1972, 87, 632. 7. Greaves, M. W., Skillen, A. W. Lancet, 1970, ii, 889. 8. Meret, S., Henkin, R. I. Clin. Chem. 1971, 17, 369. 1.