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TOTAL URINARY HYDROXYPROLINE EXCRETION AFTER ADMINISTRATION OF VITAMIN D TO HEALTHY VOLUNTEERS AND A PATIENT WITH OSTEOMALACIA
279 Glucose oxidation in whole pancreas was mannoheptulose and strongly inhibited by in clear contradistinction to the results phloridzin, obtained with mouse islets. There is thus a correlation between the effects of glucose concentration, mannoheptulose, and phloridzin on glucose oxidation in the present experiments and their effects on insulin release. The release of insulin by mouse islets in vitro as in rabbit3 or rat pancreas2 is stimulated by glucose and inhibited by mannoheptulose but not by phloridzin (E. Coll Garcia, personal communication). The present findings are therefore consistent with the idea that stimulation of insulin release by glucose may be effected by a metabolite of glucose produced in the islet cells. The possibility that an enzyme catalysing the conversion of glucose to glucose-6-phosphate is the glucoreceptor in islet cells is not established by the present study, because glucose phosphorylation is but one of a number of reactions involved in glucose oxidation. A study of glucose phosphorylating enzymes in mouse islet homogenates 11 has shown that mannoheptulose is a competitive inhibitor, thus affording an explanation for the effects of mannoheptulose in the present experiments. Glucokinase, which has been considered as a possible glucoreceptor in islet cells on account of its high Km for glucose (about 250 mg. per 100 ml.), was not detected in mouse islet homogenates. The islet-cell homogenates were found to contain a hexokinase which under some conditions has a Km for glucose of the right order (about 100 mg. per 100 ml.) to function as the
acinar tissue). unaffected by
glucoreceptor. We thank Prof. P. E. Lacy, Dr. K. W. Taylor, and Dr. E. Coll Garcia for details of their methods of islet preparation; and the British Diabetic Association and the Medical Research Council for grants which contributed to the cost of these investigations. Requests for reprints should be addressed to P. J. R., Department of Biochemistrv, Medical School. University Walk. Bristol 8.
S.
J. H. ASHCROFT B.A.
Department of Biochemistry, University of Bristol
P. J. M.D.
Cantab.
RANDLE Cantab.
TOTAL URINARY HYDROXYPROLINE EXCRETION AFTER ADMINISTRATION OF VITAMIN D TO HEALTHY VOLUNTEERS AND A PATIENT WITH OSTEOMALACIA Vitamin D in mg. dosage was given to Summary three healthy volunteers. The urinary
total hydroxyproline was measured, and did not alter. With the same dose of vitamin D the effect produced in an osteomalacic subject was different. It is suggested that the rapid increase in total hydroxyproline excretion in osteomalacia is due to temporary resorption of osteoid caused by the combined action of parathyroid hormone and increasing levels of vitamin D. INTRODUCTION
THE effect of vitamin D on the collagen of normal bone matrix is poorly understood,9 but for many years large amounts of vitamin D have been believed to cause resorption of bone mineral.10 11 In physiological doses, 8. 9. 10.
11.
Ashcroft, S. J. H., Randle, P. J. Biochem, J. (in the press). Rasmussen, H., de Luca, H. F. Ergebn. Physiol. 1963, 53, 108. Nicolaysen, R., Eeg-Larsen, N. in Vitamin and Hormones (edited by R. S. Harris G. F. Marrian, and K. V. Thimann); vol. xi, p. 29. New York, 1953. Fourman, P. Calcium Metabolism and the Bone; p. 121. Oxford, 1960.
Fig. 1-Case 1 : T.H.P. excretion, calcium and phosphorus balance, and plasma-calcium concentration.
vitamin D may be necessary for continual bone resorption and bone remodelling.ll The amount of total hydroxyproline (T.H.P.), both free and peptide-bound, in the urine is widely regarded as a reliable index of bone collagen turnover 12; it provides a new measure, albeit indirect, of the effect of vitamin D on bone. Vitamin D in curative dosage has been shown to bring about a rapid increase in T.H.P. excretion in osteomalacia of various causes, by a mechanism which is undecided.13-15 METHOD
Three healthy volunteers with presumed normal parathyroid function and normal bone structure were used as controls. Case 1, a male biochemist, aged 38 years, took vitamin D2, 2 mg. daily, for 12 days whilst on a full calcium-balance study (fig. 1). The two remaining volunteers, both of them women (case 2, aged 20 years, and case 3, aged 18 years) took pure dihydrotachysterol 2 mg. by mouth daily for 3 weeks (fig. 2). The diet was low in gelatin 12 and in case 1 the daily intake was constant throughout the balance study. For comparison, a patient (case 4) with gluten-sensitive enteropathy and osteomalacia was given the same dose of vitamin D2. This 17-year-old girl was admitted under the care of Professor Dent with a 2-year history of pain in the feet and stiffness when walking. She also had episodes of spontaneous tetany. Her childhood had been normal, and she had no diarrhoea. Her periods had started 6 months previously, and she was shorter than her sister, aged 16 years. On examination, she was sexually immature. The diagnosis of osteomalacia was based on the X-ray evidence of Looser zones, histological demonstration of excess osteoid tissue, and the biochemical Sjoerdsma, A., Udenfriend, S., Keiser, H., Le Roy, E. C. Ann. intern. Med. 1965, 63, 672. 13. Smith, D. A., Nordin, B. E. C. Proc. R. Soc. Med. 1964, 57, 868. 14. Hioco, D., Gruson, M., Ryckewaert, A., de Seze, S. Annls Biol. clin. 1967, 25, 725. 15. Smith, R., Dick, M. Clin. Sci. 1968 (in the press). 12.
280
Fig. 2-Cases 2 and 3: urinary excretion of calcium and plasma-calcium.
and of
T.H.P.,
findings shown in fig. 3. The stool-fat output was 9-3 g. per day and the intestinal biopsy specimen showed total villous atrophy. There was a gross generalised aminoaciduria. Plasmaurea was 9 mg. per 100 ml.; plasma-sodium was 141 meq. per litre; plasma-potassium was 4-3 meq. per litre; plasma-chloride was 112 meq. per litre and plasma-carbon-dioxide 22 meq. per litre. She was treated with a gluten-free diet and vitamin D2 2 mg. daily by mouth, and the biochemical response is shown in fig. 3. T.H.P. was measured in duplicate according to the method of Prockop and Udenfriend 16 on portions of 24-hour urine collections (cases 2, 3, and 4) or of 2 day pools (case 1). The variation between duplicate analyses on the same urine by this method is less than 5%.15 In the balance study on case 1, a continuous internal barium marker was used 17 and the values are, therefore, corrected for barium recovery. Plasma-calcium concentration was corrected to a specific gravity of 1-027.18 Urinary creatinine was measured byAutoanalyser ’. RESULTS
Case1 (fig. 1) After an equilibration period of 8 days, vitamin D2 2 mg. daily was begun. This produced no significant change in T.H.P. excretion, plasma-calcium concentration, or overall calcium and phosphorus balance. However, the distribution of calcium excretion between urine and faeces altered as expected, with an increase in urine calcium at the expense of faecal calcium. Cases 2 and 3 (fig. 2) After initial measurements, these volunteers took 2 mg. of pure dihydrotachysterol by mouth daily for 3 weeks. The biochemical changes were very similar in each volunteer, with a slight increase in corrected plasmacalcium concentration and a striking increase in urinary 16. 17. 18.
Prockop, D. J., Udenfriend, S. Analyt. Biochem. 1960, i, Dick, M. J. clin. Path. 1967, 20, 216. Dent, C. E. Br. med. J. 1962, ii, 1419.
Fig. 3-Case 4: biochemical changes produced by vitamin Dz 2 daily and a gluten-free diet.
calcium excretion. There excretion.
was no
mg.
significant change in the
T.H.P.
Case 4 (fig. 3)
patient, the urinary excretion of T.H.P., free hydroxyproline and phosphorus was related to that of creatinine, since some of the 24-hour urine collections were incomplete, owing to enuresis. (This was probably related to an inability to concentrate the urine associated with malabsorption and a low blood-urea J 9) There was a rapid increase in T.H.P. excretion 3 days after the vitamin D2 was begun in a dose of 2 mg. daily. This increase did not include free hydroxyproline, and coincided with a temporary rise in the excretion of urinary phosphate. The plasma-alkaline-phosphatase level tended to fall. The plasma-calcium and plasma-phosphorus concentrations showed less striking and slower increases than the T.H.P. In this
DISCUSSION
These results show that there is no increase in T.H.P. excretion in healthy persons given vitamin D (in the large doses used) and suggest that this dose of vitamin D has no effect on normal bone matrix collagen. In the balance study on case 1, the increased urinary calcium is due to increased calcium absorption from the gut unaided by mobilisation of bone mineral. In the other two volunteers in whom
228.
19.
Klahr, S., Tripathy, K., Garcia, F. T., Mayoral, L. G., Ghitis, J., Bolanos, O. Am. J. med. 1967, 43, 84.
281 vitamin D (in the form of dihydrotachysterol) was taken for a longer period, there was pronounced hypercalcuria; again this was probably due to increased calcium absorption from the gut, and the measurements of T.H.P. excretion do not indicate bone resorption. These findings contrast strongly with those found in osteomalacia, as shown in case 4 and in patients described elsewhere.15 Mellanby20 showed nearly 20 years ago that vitamin D had a direct effect on the structure of osteomalacic bone in puppies, seemingly independent of its effect on calcium absorption from the gut. He studied the radiological appearances, histology, and the ratio of total mineral content to dried fat-free organic matter, but made no measurement of bone-collagen change. The difference in the T.H.P. response to the same dose of vitamin D in healthy and osteomalacic subjects may be due to difference either in degree of parathyroid activity or in bone-matrix collagen. In the volunteers, parathyroid function is presumably normal. In osteomalacia, however, there is a variable degree of secondary parathyroid overactivity ; in case 4 this is suggested by subperiosteal erosions of the phalanges on X-ray, by resorption cavities in addition to excess osteoid on microscopy of bone and more ambiguously by the low plasma-phosphorus. Osteomalacic bone-collagen may differ from normal bonecollagen, but there is no evidence for this. When vitamin D is given to patients with osteomalacia, 20.
Mellanby,
E.
J. Physiol. (Lond.) 1949, 109, 488.
R. S. is a Senior Wellcome Research Fellow and acknowledges with thanks financial and technical assistance from the Wellcome Trust. We are grateful to the volunteers in this investigation, to Prof. C. E. Dent for his advice, and to Miss Margaret Bishop for technical assistance. Requests for reprints should be addressed to R. S. Medical Unit, London W.C.1 21.
ROGER SMITH Cantab., M.R.C.P. MATTHEW DICK B.SC. Glasg.
M.A. M.D.
University College Hospital Medical School,
22.
Hypothesis
Goldhaber, P. in The Parathyroid Glands (edited by P. J. Gaillard, R. V. Talmage, and A. M. Budy); p. 153. Chicago, 1965. Evanson, J. M. Clin. Sci. 1966, 31, 63.
by foreign antigenic determinants; and amplification of A number of more detailed, and hence more controversial, postulates will be made here. These 6 are (a) that randomisation occurs in the thymus,5 (b) that the units involved are single cells,78 (c) that such cells are released from the thymus to seed other tissues,9 10 and (d) that at some stage during this process self-recognition occurs. A possible mechanism by which self-recognition this response.
THE LIQUID SCINTILLATION COUNTER AS AN ANALOGY FOR THE DISTINCTION BETWEEN " SELF " AND " NOT-SELF " IN IMMUNOLOGICAL SYSTEMS
The natural selection theory of immunity of Jerne, with its emphasis on natural antibody, is combined with the clonal selection theory of Burnet, with its emphasis on cells, to produce a simple " two site " hypothesis of the mechanism of immune selfrecognition in vivo. An analogy is made with a similar process occurring in liquid scintillation counters containing two photocells and a coincidence circuit.
Summary
INTRODUCTION
I EXPLORE here the extent to which the principles of known process in a non-biological system can be applied to the biological problem of immune self-recognition in vivo. The result is a scheme of interrelationships between various cellular and humoral factors which, although it rests on tenuous experimental evidence, seems to be not too inconsistent with the currently accepted facts of a
immunology. Selective theories of immunity 1-4 have four main elements: a randomisation process by which a wide spectrum of units, each able to direct the formation of a specific antibody, are established; a mechanism forbidding immune reaction with " self " and permitting recognition of foreign (" not-self ") antigenic determinants ; activation of the synthesis of specific antibody 1. Jerne, N. K. Proc. natn. Acad. Sci., Wash. 1955, 41, 849. 2. Burnet, F. M. The Clonal Selection Theory of Acquired 1959. 3. Lederberg, J. Science, N.Y. 1959, 129, 1649. 4. Eisen, H. N., Karush, F. Nature, Lond. 1964, 202, 677.
vitamin-D concentration increases in the presence of functionally overactive parathyroids. Since there is experimental 21 and clinical 22 evidence that parathyroid hormone requires an adequate concentration of vitamin D for its action on bone, we suggest that the rapid increase in T.H.P. excretion is due to temporary resorption of osteoid caused by the combined synergistic action of increasing levels of vitamin D and parathyroid hormone. There is no evidence from our results that increased synthesis of collagen occurs at this stage, and in case 4 the plasmaalkaline-phosphatase (which reflects osteoblastic activity) does not increase with increasing T.H.P. Further data to support this hypothesis are being collected. Analysis of the separate hydroxyproline peptides produced in the urine during this increase in T.H.P. could give insight into the methods of breakdown of bone-collagen.
Immunity.
could occur in vivo will be derived from a consideration of an analogous process occurring in modern liquid scintillation counters. Insofar as activation is closely linked with the postulated recognition process it will also be discussed. Neither the mechanism of randomisation nor amplification, will be considered here. LIQUID
SCINTILLATION COUNTING
Radioactivity in a scintillation solution emits pulses of light energy which may be detected by a photocell 11 which converts light energy into electrical energy. In theory, simply connecting the photocell to a counting meter should provide a record of the disintegrations occurring in the solution, and thus of the amount of radiopresent. In practice, however, the background activity " noise " within the photocell seriously reduces the sensitivity of the method. The counting meter needs to distinguish between pulses of energy originating within the scintillation solution (" not-self "), and those originating within the photocell (" self "). The difficulty is overcome by using a coincidence circuit in which the scintillation solution is looked at, not Claman, H. N., Talmage, D. W. Science, N.Y. 1963, 141, 1193. 6. Cross, A. M., Leuchars, E., Miller, J. F. A. P. J. exp. Med. 1964, 119, 5.
837.
Nossal, J. G. V., Szenberg, A., Ada, G. L. G., Austin, C. M. ibid. 1963, 119, 485. 8. Friedman, H. P. Experientia, Basel, 1964, 20, 564. 9. Nossal, J. G. V. Ann. N. Y. Acad. Sci. 1964, 120, 171. 10. Murray, R. G., Woods, P. A. Anat. Rec. 1964, 150, 113. 11. Bell, C. G., Hayes, F. N. (editors). Liquid Scintillation Counting. London, 1958. 7.
acinar tissue). unaffected by
glucoreceptor. We thank Prof. P. E. Lacy, Dr. K. W. Taylor, and Dr. E. Coll Garcia for details of their methods of islet preparation; and the British Diabetic Association and the Medical Research Council for grants which contributed to the cost of these investigations. Requests for reprints should be addressed to P. J. R., Department of Biochemistrv, Medical School. University Walk. Bristol 8.
S.
J. H. ASHCROFT B.A.
Department of Biochemistry, University of Bristol
P. J. M.D.
Cantab.
RANDLE Cantab.
TOTAL URINARY HYDROXYPROLINE EXCRETION AFTER ADMINISTRATION OF VITAMIN D TO HEALTHY VOLUNTEERS AND A PATIENT WITH OSTEOMALACIA Vitamin D in mg. dosage was given to Summary three healthy volunteers. The urinary
total hydroxyproline was measured, and did not alter. With the same dose of vitamin D the effect produced in an osteomalacic subject was different. It is suggested that the rapid increase in total hydroxyproline excretion in osteomalacia is due to temporary resorption of osteoid caused by the combined action of parathyroid hormone and increasing levels of vitamin D. INTRODUCTION
THE effect of vitamin D on the collagen of normal bone matrix is poorly understood,9 but for many years large amounts of vitamin D have been believed to cause resorption of bone mineral.10 11 In physiological doses, 8. 9. 10.
11.
Ashcroft, S. J. H., Randle, P. J. Biochem, J. (in the press). Rasmussen, H., de Luca, H. F. Ergebn. Physiol. 1963, 53, 108. Nicolaysen, R., Eeg-Larsen, N. in Vitamin and Hormones (edited by R. S. Harris G. F. Marrian, and K. V. Thimann); vol. xi, p. 29. New York, 1953. Fourman, P. Calcium Metabolism and the Bone; p. 121. Oxford, 1960.
Fig. 1-Case 1 : T.H.P. excretion, calcium and phosphorus balance, and plasma-calcium concentration.
vitamin D may be necessary for continual bone resorption and bone remodelling.ll The amount of total hydroxyproline (T.H.P.), both free and peptide-bound, in the urine is widely regarded as a reliable index of bone collagen turnover 12; it provides a new measure, albeit indirect, of the effect of vitamin D on bone. Vitamin D in curative dosage has been shown to bring about a rapid increase in T.H.P. excretion in osteomalacia of various causes, by a mechanism which is undecided.13-15 METHOD
Three healthy volunteers with presumed normal parathyroid function and normal bone structure were used as controls. Case 1, a male biochemist, aged 38 years, took vitamin D2, 2 mg. daily, for 12 days whilst on a full calcium-balance study (fig. 1). The two remaining volunteers, both of them women (case 2, aged 20 years, and case 3, aged 18 years) took pure dihydrotachysterol 2 mg. by mouth daily for 3 weeks (fig. 2). The diet was low in gelatin 12 and in case 1 the daily intake was constant throughout the balance study. For comparison, a patient (case 4) with gluten-sensitive enteropathy and osteomalacia was given the same dose of vitamin D2. This 17-year-old girl was admitted under the care of Professor Dent with a 2-year history of pain in the feet and stiffness when walking. She also had episodes of spontaneous tetany. Her childhood had been normal, and she had no diarrhoea. Her periods had started 6 months previously, and she was shorter than her sister, aged 16 years. On examination, she was sexually immature. The diagnosis of osteomalacia was based on the X-ray evidence of Looser zones, histological demonstration of excess osteoid tissue, and the biochemical Sjoerdsma, A., Udenfriend, S., Keiser, H., Le Roy, E. C. Ann. intern. Med. 1965, 63, 672. 13. Smith, D. A., Nordin, B. E. C. Proc. R. Soc. Med. 1964, 57, 868. 14. Hioco, D., Gruson, M., Ryckewaert, A., de Seze, S. Annls Biol. clin. 1967, 25, 725. 15. Smith, R., Dick, M. Clin. Sci. 1968 (in the press). 12.
280
Fig. 2-Cases 2 and 3: urinary excretion of calcium and plasma-calcium.
and of
T.H.P.,
findings shown in fig. 3. The stool-fat output was 9-3 g. per day and the intestinal biopsy specimen showed total villous atrophy. There was a gross generalised aminoaciduria. Plasmaurea was 9 mg. per 100 ml.; plasma-sodium was 141 meq. per litre; plasma-potassium was 4-3 meq. per litre; plasma-chloride was 112 meq. per litre and plasma-carbon-dioxide 22 meq. per litre. She was treated with a gluten-free diet and vitamin D2 2 mg. daily by mouth, and the biochemical response is shown in fig. 3. T.H.P. was measured in duplicate according to the method of Prockop and Udenfriend 16 on portions of 24-hour urine collections (cases 2, 3, and 4) or of 2 day pools (case 1). The variation between duplicate analyses on the same urine by this method is less than 5%.15 In the balance study on case 1, a continuous internal barium marker was used 17 and the values are, therefore, corrected for barium recovery. Plasma-calcium concentration was corrected to a specific gravity of 1-027.18 Urinary creatinine was measured byAutoanalyser ’. RESULTS
Case1 (fig. 1) After an equilibration period of 8 days, vitamin D2 2 mg. daily was begun. This produced no significant change in T.H.P. excretion, plasma-calcium concentration, or overall calcium and phosphorus balance. However, the distribution of calcium excretion between urine and faeces altered as expected, with an increase in urine calcium at the expense of faecal calcium. Cases 2 and 3 (fig. 2) After initial measurements, these volunteers took 2 mg. of pure dihydrotachysterol by mouth daily for 3 weeks. The biochemical changes were very similar in each volunteer, with a slight increase in corrected plasmacalcium concentration and a striking increase in urinary 16. 17. 18.
Prockop, D. J., Udenfriend, S. Analyt. Biochem. 1960, i, Dick, M. J. clin. Path. 1967, 20, 216. Dent, C. E. Br. med. J. 1962, ii, 1419.
Fig. 3-Case 4: biochemical changes produced by vitamin Dz 2 daily and a gluten-free diet.
calcium excretion. There excretion.
was no
mg.
significant change in the
T.H.P.
Case 4 (fig. 3)
patient, the urinary excretion of T.H.P., free hydroxyproline and phosphorus was related to that of creatinine, since some of the 24-hour urine collections were incomplete, owing to enuresis. (This was probably related to an inability to concentrate the urine associated with malabsorption and a low blood-urea J 9) There was a rapid increase in T.H.P. excretion 3 days after the vitamin D2 was begun in a dose of 2 mg. daily. This increase did not include free hydroxyproline, and coincided with a temporary rise in the excretion of urinary phosphate. The plasma-alkaline-phosphatase level tended to fall. The plasma-calcium and plasma-phosphorus concentrations showed less striking and slower increases than the T.H.P. In this
DISCUSSION
These results show that there is no increase in T.H.P. excretion in healthy persons given vitamin D (in the large doses used) and suggest that this dose of vitamin D has no effect on normal bone matrix collagen. In the balance study on case 1, the increased urinary calcium is due to increased calcium absorption from the gut unaided by mobilisation of bone mineral. In the other two volunteers in whom
228.
19.
Klahr, S., Tripathy, K., Garcia, F. T., Mayoral, L. G., Ghitis, J., Bolanos, O. Am. J. med. 1967, 43, 84.
281 vitamin D (in the form of dihydrotachysterol) was taken for a longer period, there was pronounced hypercalcuria; again this was probably due to increased calcium absorption from the gut, and the measurements of T.H.P. excretion do not indicate bone resorption. These findings contrast strongly with those found in osteomalacia, as shown in case 4 and in patients described elsewhere.15 Mellanby20 showed nearly 20 years ago that vitamin D had a direct effect on the structure of osteomalacic bone in puppies, seemingly independent of its effect on calcium absorption from the gut. He studied the radiological appearances, histology, and the ratio of total mineral content to dried fat-free organic matter, but made no measurement of bone-collagen change. The difference in the T.H.P. response to the same dose of vitamin D in healthy and osteomalacic subjects may be due to difference either in degree of parathyroid activity or in bone-matrix collagen. In the volunteers, parathyroid function is presumably normal. In osteomalacia, however, there is a variable degree of secondary parathyroid overactivity ; in case 4 this is suggested by subperiosteal erosions of the phalanges on X-ray, by resorption cavities in addition to excess osteoid on microscopy of bone and more ambiguously by the low plasma-phosphorus. Osteomalacic bone-collagen may differ from normal bonecollagen, but there is no evidence for this. When vitamin D is given to patients with osteomalacia, 20.
Mellanby,
E.
J. Physiol. (Lond.) 1949, 109, 488.
R. S. is a Senior Wellcome Research Fellow and acknowledges with thanks financial and technical assistance from the Wellcome Trust. We are grateful to the volunteers in this investigation, to Prof. C. E. Dent for his advice, and to Miss Margaret Bishop for technical assistance. Requests for reprints should be addressed to R. S. Medical Unit, London W.C.1 21.
ROGER SMITH Cantab., M.R.C.P. MATTHEW DICK B.SC. Glasg.
M.A. M.D.
University College Hospital Medical School,
22.
Hypothesis
Goldhaber, P. in The Parathyroid Glands (edited by P. J. Gaillard, R. V. Talmage, and A. M. Budy); p. 153. Chicago, 1965. Evanson, J. M. Clin. Sci. 1966, 31, 63.
by foreign antigenic determinants; and amplification of A number of more detailed, and hence more controversial, postulates will be made here. These 6 are (a) that randomisation occurs in the thymus,5 (b) that the units involved are single cells,78 (c) that such cells are released from the thymus to seed other tissues,9 10 and (d) that at some stage during this process self-recognition occurs. A possible mechanism by which self-recognition this response.
THE LIQUID SCINTILLATION COUNTER AS AN ANALOGY FOR THE DISTINCTION BETWEEN " SELF " AND " NOT-SELF " IN IMMUNOLOGICAL SYSTEMS
The natural selection theory of immunity of Jerne, with its emphasis on natural antibody, is combined with the clonal selection theory of Burnet, with its emphasis on cells, to produce a simple " two site " hypothesis of the mechanism of immune selfrecognition in vivo. An analogy is made with a similar process occurring in liquid scintillation counters containing two photocells and a coincidence circuit.
Summary
INTRODUCTION
I EXPLORE here the extent to which the principles of known process in a non-biological system can be applied to the biological problem of immune self-recognition in vivo. The result is a scheme of interrelationships between various cellular and humoral factors which, although it rests on tenuous experimental evidence, seems to be not too inconsistent with the currently accepted facts of a
immunology. Selective theories of immunity 1-4 have four main elements: a randomisation process by which a wide spectrum of units, each able to direct the formation of a specific antibody, are established; a mechanism forbidding immune reaction with " self " and permitting recognition of foreign (" not-self ") antigenic determinants ; activation of the synthesis of specific antibody 1. Jerne, N. K. Proc. natn. Acad. Sci., Wash. 1955, 41, 849. 2. Burnet, F. M. The Clonal Selection Theory of Acquired 1959. 3. Lederberg, J. Science, N.Y. 1959, 129, 1649. 4. Eisen, H. N., Karush, F. Nature, Lond. 1964, 202, 677.
vitamin-D concentration increases in the presence of functionally overactive parathyroids. Since there is experimental 21 and clinical 22 evidence that parathyroid hormone requires an adequate concentration of vitamin D for its action on bone, we suggest that the rapid increase in T.H.P. excretion is due to temporary resorption of osteoid caused by the combined synergistic action of increasing levels of vitamin D and parathyroid hormone. There is no evidence from our results that increased synthesis of collagen occurs at this stage, and in case 4 the plasmaalkaline-phosphatase (which reflects osteoblastic activity) does not increase with increasing T.H.P. Further data to support this hypothesis are being collected. Analysis of the separate hydroxyproline peptides produced in the urine during this increase in T.H.P. could give insight into the methods of breakdown of bone-collagen.
Immunity.
could occur in vivo will be derived from a consideration of an analogous process occurring in modern liquid scintillation counters. Insofar as activation is closely linked with the postulated recognition process it will also be discussed. Neither the mechanism of randomisation nor amplification, will be considered here. LIQUID
SCINTILLATION COUNTING
Radioactivity in a scintillation solution emits pulses of light energy which may be detected by a photocell 11 which converts light energy into electrical energy. In theory, simply connecting the photocell to a counting meter should provide a record of the disintegrations occurring in the solution, and thus of the amount of radiopresent. In practice, however, the background activity " noise " within the photocell seriously reduces the sensitivity of the method. The counting meter needs to distinguish between pulses of energy originating within the scintillation solution (" not-self "), and those originating within the photocell (" self "). The difficulty is overcome by using a coincidence circuit in which the scintillation solution is looked at, not Claman, H. N., Talmage, D. W. Science, N.Y. 1963, 141, 1193. 6. Cross, A. M., Leuchars, E., Miller, J. F. A. P. J. exp. Med. 1964, 119, 5.
837.
Nossal, J. G. V., Szenberg, A., Ada, G. L. G., Austin, C. M. ibid. 1963, 119, 485. 8. Friedman, H. P. Experientia, Basel, 1964, 20, 564. 9. Nossal, J. G. V. Ann. N. Y. Acad. Sci. 1964, 120, 171. 10. Murray, R. G., Woods, P. A. Anat. Rec. 1964, 150, 113. 11. Bell, C. G., Hayes, F. N. (editors). Liquid Scintillation Counting. London, 1958. 7.