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URINARY ALBUMIN EXCRETION AND DIABETES MELLITUS
1155 the homologous antigen before the test abolished heemagglutination or caused considerable decrease in the titre. Absorption with AB Rh-positive human red-cells made no difference to the final titre. Furthermore, significant antibody levels were not shown with liver, colon, and skin sensitised red cells. DISCUSSION
The agglutination titre of the healthy controls and of the patients with systemic disease did not exceed 1/32. But 7% (2/30) of the non-ulcerating mouth lesions, and 10% (4/40) of the ulcerating lesions gave titres of over 1/32. This proportion rose to 75% (45/60) in patients with aphthous ulcers. Thus, serum from patients with aphthous ulcers caused a specific agglutination of cells that had been coated with an extract of oral mucosa in higher titre and greater proportion than did those of controls and of other diseases. Moreover, no appreciable difference could be detected between the other ulcerating The evidence, therefore, and non-ulcerating lesions. this is more that than an suggests immunological reaction to non-specific mucosal damage. The significance of these findings cannot be fully assessed until the results are known of a variety of immunological tests which are now being carried out. It seems, however, that the part played by an autoimmune reaction or an antibody cross-reacting with an infective agent and oral mucosa deserves further consideration. I am greatly indebted to Prof. M. A. Rushton for his helpful advice and encouragement, and for allowing me to investigate his patients; to Dr. M. H. Lessof for his guidance and generous advice; and to Mr. C. Theophanides for his valuable technical assistance. My thanks are also due to Dr. J. R. Trounce and the members of the interdepart-
from three groups of people who were identified in the survey by their blood-sugar 2 hours after 50 g.
glucose by mouth, as
follows:
1. Those with a 2-hour blood-sugar above 200 mg. per 100 ml. were desig-
nated " diabetics ". 2. Those with a 2-hour blood-sugar between 120 and 200 mg. per 100 ml. were "
designated Fig. 1-Frequency-distribution
borderline diabetics ". 3. Those with a 2-hour blood-sugar
histograms showing numbers (ordinate) of subjects in each 10-year age-group for the sexes separately, divided into diabetics, borderline diabetics, and normal controls.
were designated normal controls ". The reasons for and purpose of this grouping have been discussed elsewhere.5-7 Following the survev, people from the above three groups were
below 120 mg. per 100 ml.
subjected to a follow-up examination, including a further " 2hour bloodsugar ", and the collection of a timed and measured 2hour urineThe output. albumin content was estimated by a simple and sensitive radio-
mental laboratory committee for permission to use the facilities of the interdepartmental laboratory; and to Prof. R. E. Emslie, Dr. R. A. Cawson, Dr. L. Forman, and Mr. D. Neill for allowing me access to their patients.
Department of Dental Medicine, Guy’s Hospital Medical School, London, S.E.1
THOMAS LEHNER B.D.S. Lond., F.D.S.
M.B.,
URINARY ALBUMIN EXCRETION AND DIABETES MELLITUS OF the clinical features which characterise diabetic nephropathy, proteinuria has been recognised as the first clinical hint.1 Light-microscopic examinations of renal tissue from longstanding diabetics shows gross alterations in the basement membrane of the glomerular capillaries.22 More recent studies with the electron microscope have shown quite distinct changes of a similar nature at a very early stage of the diabetes, before any clinical evidence of renal disease is apparent, and even in so-called prediabetes in some cases.34 Proteinuria was not found in these early cases using the ordinary clinical tests; these are, however, inadequate to measure small amounts of protein in the urine, and even a tenfold to twentyfold increase of urinary protein excretion over the normal would pass undetected. Thus it seemed possible that a counterpart of these very early pathological changes might be found in large but clinically undetectable increases in the rate of urinary protein excretion. This possibility was examined in a sample of people studied in the Bedford diabetes survey.5-’
immunoassay method, specific for albumin, 2-Frequency-distribution of urinary albumin concentrations (mg. per 100 ml.) in the three groups studied. The concentration range 0 to 1 mg. per 100 ml., comprising more than half the observations, has been subdivided into four 0’25 mg. per 100 ml. steps. Fig.
2. 3. 4.
5. 6. 7.
dom the
ran-
sample of three
groups. A total of 295 urine
samples were assayed: 55
compositions of the borderline diabetic and control
Urine samples examined in the present study were collected Gellman, D. D., Pirani, C. L., Soothill, J. F., Muchrche, R. C., Kark, R. M. Medicine, 1959, 38, 321. Kimmelstiel, P., Wilson, C. Amer. J. Path. 1936, 12, 83. Sabour, M. S., MacDonald, M. K., Robson, I. S. Diabetes, 1962, 11, 291. Camerini-Davalos, R. A., Chaulfield, J. B., Rees, S. B., LozanoCastaneda, O., Naldjian, S., Marble, A. ibid. 1963, 12, 508. Sharp, C. L. Proc. R. Soc. Med. 1964, 57, 193. Butterfield, W. J. H. ibid. p. 196. Keen, H. ibid. p. 200.
frozen ali-
quots of a
from diabetics ", 112 from " borderline diabetics ", and 128 from " normal controls ". The age and sex distribution of these subjects is shown in The fig. 1.
MATERIAL AND METHODS
1.
on
Fig.
3 - Frequency distribution
of 2-hour urinary-albumin excretion-rates (mg. per 2 hr.), calculated from albumin concentration and 2-hour urine-volume. The range 0-1 mg. per 2 hours has again been subdivided into four equal segments.
groups
are
very
similar, though the diabetics contain an excess of older
people.
1156 DISCUSSION
OUTLINE OF ALBUMIN-ASSAY METHOD
The immunoassay method employed has been described elsewhere. In outline, a fixed quantity of human albumin labelled with 125I is added to a measured volume of the test urine (0-1 ml.). To this mixture is added antihuman-albumin serum raised in the guineapig; and, after equilibration, albumin-
antibody complexes are precipitated by an antiguineapigglobulin serum raised in the rabbit. The radioactivity found in the precipitate is related to the quantity of albumin in the test urine, which is derived by interpolation into a standard curve. RESULTS
.
The distribution of the urinary-albumin concentrations in each of the three groups is shown in fig. 2. The distributions are very asymetrical, and of the type; more than half the urine samples had an albumin concentration of 1 mg. per 100 ml. or
less. The
frequency-distribution of the 2-hour albumin excretionvalues, calculated from the albumin concentration and the 2-hour volume, is shown in fig. 3. This distribution is also very asymetrical, resembling that of the albumin rate
concentration. Differences of urinary albumin excretion-rates among the groups cannot be assessed by statistical tests based upon the assumption of normally distributed variates. To make the comparison, all the albumin excretion-rates were ranked by value and then divided into six equal segments. The percentage representation ofeach of the three groups-diabetics, border-
The increased urinary albumin excretion demonstrated in the newly discovered and borderline diabetics in Bedford may well be due to an increased permeability of the glomerular basement membrane very early in the clinical course of the condition, and reflecting the morphological changes mentioned earlier. Alternative explanations must be considered, however. Urinary-albumin excretion-rates will depend not only upon the rate of entry of albumin into the glomerular filtrate but also upon the rate of its reabsorption in the renal tubules. A diminution in tubular albumin reabsorption may occur as a result of some abnormality of the tubular epithelium or if reabsorption is competitively hindered by the presence of other small molecular weight proteins; there is no evidence at present supporting these possibilities in early diabetes. Loss of plasma-proteins into the urinary tract at sites other than the renal glomerulus would also give rise to increased albuminuria. Such losses could arise from inflammation or haemorrhage anywhere in the urinary tract and, in this present study, the intervention of such factors cannot be ruled out. But casts and cells per se in the urine do not contribute to the estimated albumin content measured as described (unpublished observations). If we assume that the increased albuminuria associated with raised blood-sugar levels is a manifestation of a specific " diabetic " change in glomerular permeability, then the evidence of this study suggests that the grade of change may be quantitatively related to the degree of hyperglycsemia. Such a relationship would be most simply explained by a direct effect of the circulating glucose level upon the structure or function of the glomerular basement membrane, although it is also clearly possible that bloodglucose levels and basement-membrane behaviour might both be influenced by a common factor. Too little information is at present available to make any more than speculative attempts to differentiate between these
possibilities. Fig. 4-Percentage representation of the three excretion-rate hexile (see text). "
groups in each
"
line diabetics,and normal controls-in each " hexile " is shown in fig. 4. There is a clear tendency for diabetics to be better represented in the higher excretion-rate categories, with the reverse for the normal controls. Borderline diabetics occupy an intermediate position. On the basis of this classification a 6 x 3 contingency table was constructed and a 1.2 test showed that the different representation of the groups in each hexile was very unlikely to have arisen by chance (P==0’0006). The table shows a further comparison of mean albumin excretion-rates when subjects were classified according to their 2-hour blood-sugar not only at the survey but also at their visit to the follow-up clinic. (D=200 mg. or more per 100 ml., B =120-199 mg. per 100 ml., N=119 mg. per 100 ml. or less.) It can be seen that the calculated albumin excretion-rate shows a stepwise decline from the " most " to the " least " hyper-
glycaemic
group:
It remains to be seen whether these " subclinical increases in albuminuria in the diabetic are correlated with morphological changes in the glomeruli or with subsequent clinical development of renal disease, and whether treatment will influence these events. SUMMARY
Using a sensitive and specific method for measuring albumin in the urine of three groups consisting of early diabetics, " borderline diabetics ", and normal people, a gradation in the rate of urinary-albumin excretion was found. Diabetics excreted the most and normals the least albumin of the three groups. This gradation in the rate of albumin excretion occurred at concentrations undetectable by ordinary clinical tests. The increased albuminuria may be related to the glomerular basement-membrane changes reported by other workers, but further correlative studies are
required. This work was supported by a research grant from the British Diabetic Association and by a U.S. Public Health Service Grant. Acknowledgments are also due to Mr. N. Veall for preparing the 1251-labelled albumin, and to Mr. M. Field, Miss A. Southon, and Miss J. Williams for technical assistance.
HARRY KEEN Lond, M.R.C.P. C. CHLOUVERAKIS M.B.
8.
Keen, H., Chlouverakis, C. Lancet, 1963, ii, 913.
Department of Medicine Guy’s Hospital, London, S.E.1
M.D.
Athens
The agglutination titre of the healthy controls and of the patients with systemic disease did not exceed 1/32. But 7% (2/30) of the non-ulcerating mouth lesions, and 10% (4/40) of the ulcerating lesions gave titres of over 1/32. This proportion rose to 75% (45/60) in patients with aphthous ulcers. Thus, serum from patients with aphthous ulcers caused a specific agglutination of cells that had been coated with an extract of oral mucosa in higher titre and greater proportion than did those of controls and of other diseases. Moreover, no appreciable difference could be detected between the other ulcerating The evidence, therefore, and non-ulcerating lesions. this is more that than an suggests immunological reaction to non-specific mucosal damage. The significance of these findings cannot be fully assessed until the results are known of a variety of immunological tests which are now being carried out. It seems, however, that the part played by an autoimmune reaction or an antibody cross-reacting with an infective agent and oral mucosa deserves further consideration. I am greatly indebted to Prof. M. A. Rushton for his helpful advice and encouragement, and for allowing me to investigate his patients; to Dr. M. H. Lessof for his guidance and generous advice; and to Mr. C. Theophanides for his valuable technical assistance. My thanks are also due to Dr. J. R. Trounce and the members of the interdepart-
from three groups of people who were identified in the survey by their blood-sugar 2 hours after 50 g.
glucose by mouth, as
follows:
1. Those with a 2-hour blood-sugar above 200 mg. per 100 ml. were desig-
nated " diabetics ". 2. Those with a 2-hour blood-sugar between 120 and 200 mg. per 100 ml. were "
designated Fig. 1-Frequency-distribution
borderline diabetics ". 3. Those with a 2-hour blood-sugar
histograms showing numbers (ordinate) of subjects in each 10-year age-group for the sexes separately, divided into diabetics, borderline diabetics, and normal controls.
were designated normal controls ". The reasons for and purpose of this grouping have been discussed elsewhere.5-7 Following the survev, people from the above three groups were
below 120 mg. per 100 ml.
subjected to a follow-up examination, including a further " 2hour bloodsugar ", and the collection of a timed and measured 2hour urineThe output. albumin content was estimated by a simple and sensitive radio-
mental laboratory committee for permission to use the facilities of the interdepartmental laboratory; and to Prof. R. E. Emslie, Dr. R. A. Cawson, Dr. L. Forman, and Mr. D. Neill for allowing me access to their patients.
Department of Dental Medicine, Guy’s Hospital Medical School, London, S.E.1
THOMAS LEHNER B.D.S. Lond., F.D.S.
M.B.,
URINARY ALBUMIN EXCRETION AND DIABETES MELLITUS OF the clinical features which characterise diabetic nephropathy, proteinuria has been recognised as the first clinical hint.1 Light-microscopic examinations of renal tissue from longstanding diabetics shows gross alterations in the basement membrane of the glomerular capillaries.22 More recent studies with the electron microscope have shown quite distinct changes of a similar nature at a very early stage of the diabetes, before any clinical evidence of renal disease is apparent, and even in so-called prediabetes in some cases.34 Proteinuria was not found in these early cases using the ordinary clinical tests; these are, however, inadequate to measure small amounts of protein in the urine, and even a tenfold to twentyfold increase of urinary protein excretion over the normal would pass undetected. Thus it seemed possible that a counterpart of these very early pathological changes might be found in large but clinically undetectable increases in the rate of urinary protein excretion. This possibility was examined in a sample of people studied in the Bedford diabetes survey.5-’
immunoassay method, specific for albumin, 2-Frequency-distribution of urinary albumin concentrations (mg. per 100 ml.) in the three groups studied. The concentration range 0 to 1 mg. per 100 ml., comprising more than half the observations, has been subdivided into four 0’25 mg. per 100 ml. steps. Fig.
2. 3. 4.
5. 6. 7.
dom the
ran-
sample of three
groups. A total of 295 urine
samples were assayed: 55
compositions of the borderline diabetic and control
Urine samples examined in the present study were collected Gellman, D. D., Pirani, C. L., Soothill, J. F., Muchrche, R. C., Kark, R. M. Medicine, 1959, 38, 321. Kimmelstiel, P., Wilson, C. Amer. J. Path. 1936, 12, 83. Sabour, M. S., MacDonald, M. K., Robson, I. S. Diabetes, 1962, 11, 291. Camerini-Davalos, R. A., Chaulfield, J. B., Rees, S. B., LozanoCastaneda, O., Naldjian, S., Marble, A. ibid. 1963, 12, 508. Sharp, C. L. Proc. R. Soc. Med. 1964, 57, 193. Butterfield, W. J. H. ibid. p. 196. Keen, H. ibid. p. 200.
frozen ali-
quots of a
from diabetics ", 112 from " borderline diabetics ", and 128 from " normal controls ". The age and sex distribution of these subjects is shown in The fig. 1.
MATERIAL AND METHODS
1.
on
Fig.
3 - Frequency distribution
of 2-hour urinary-albumin excretion-rates (mg. per 2 hr.), calculated from albumin concentration and 2-hour urine-volume. The range 0-1 mg. per 2 hours has again been subdivided into four equal segments.
groups
are
very
similar, though the diabetics contain an excess of older
people.
1156 DISCUSSION
OUTLINE OF ALBUMIN-ASSAY METHOD
The immunoassay method employed has been described elsewhere. In outline, a fixed quantity of human albumin labelled with 125I is added to a measured volume of the test urine (0-1 ml.). To this mixture is added antihuman-albumin serum raised in the guineapig; and, after equilibration, albumin-
antibody complexes are precipitated by an antiguineapigglobulin serum raised in the rabbit. The radioactivity found in the precipitate is related to the quantity of albumin in the test urine, which is derived by interpolation into a standard curve. RESULTS
.
The distribution of the urinary-albumin concentrations in each of the three groups is shown in fig. 2. The distributions are very asymetrical, and of the type; more than half the urine samples had an albumin concentration of 1 mg. per 100 ml. or
less. The
frequency-distribution of the 2-hour albumin excretionvalues, calculated from the albumin concentration and the 2-hour volume, is shown in fig. 3. This distribution is also very asymetrical, resembling that of the albumin rate
concentration. Differences of urinary albumin excretion-rates among the groups cannot be assessed by statistical tests based upon the assumption of normally distributed variates. To make the comparison, all the albumin excretion-rates were ranked by value and then divided into six equal segments. The percentage representation ofeach of the three groups-diabetics, border-
The increased urinary albumin excretion demonstrated in the newly discovered and borderline diabetics in Bedford may well be due to an increased permeability of the glomerular basement membrane very early in the clinical course of the condition, and reflecting the morphological changes mentioned earlier. Alternative explanations must be considered, however. Urinary-albumin excretion-rates will depend not only upon the rate of entry of albumin into the glomerular filtrate but also upon the rate of its reabsorption in the renal tubules. A diminution in tubular albumin reabsorption may occur as a result of some abnormality of the tubular epithelium or if reabsorption is competitively hindered by the presence of other small molecular weight proteins; there is no evidence at present supporting these possibilities in early diabetes. Loss of plasma-proteins into the urinary tract at sites other than the renal glomerulus would also give rise to increased albuminuria. Such losses could arise from inflammation or haemorrhage anywhere in the urinary tract and, in this present study, the intervention of such factors cannot be ruled out. But casts and cells per se in the urine do not contribute to the estimated albumin content measured as described (unpublished observations). If we assume that the increased albuminuria associated with raised blood-sugar levels is a manifestation of a specific " diabetic " change in glomerular permeability, then the evidence of this study suggests that the grade of change may be quantitatively related to the degree of hyperglycsemia. Such a relationship would be most simply explained by a direct effect of the circulating glucose level upon the structure or function of the glomerular basement membrane, although it is also clearly possible that bloodglucose levels and basement-membrane behaviour might both be influenced by a common factor. Too little information is at present available to make any more than speculative attempts to differentiate between these
possibilities. Fig. 4-Percentage representation of the three excretion-rate hexile (see text). "
groups in each
"
line diabetics,and normal controls-in each " hexile " is shown in fig. 4. There is a clear tendency for diabetics to be better represented in the higher excretion-rate categories, with the reverse for the normal controls. Borderline diabetics occupy an intermediate position. On the basis of this classification a 6 x 3 contingency table was constructed and a 1.2 test showed that the different representation of the groups in each hexile was very unlikely to have arisen by chance (P==0’0006). The table shows a further comparison of mean albumin excretion-rates when subjects were classified according to their 2-hour blood-sugar not only at the survey but also at their visit to the follow-up clinic. (D=200 mg. or more per 100 ml., B =120-199 mg. per 100 ml., N=119 mg. per 100 ml. or less.) It can be seen that the calculated albumin excretion-rate shows a stepwise decline from the " most " to the " least " hyper-
glycaemic
group:
It remains to be seen whether these " subclinical increases in albuminuria in the diabetic are correlated with morphological changes in the glomeruli or with subsequent clinical development of renal disease, and whether treatment will influence these events. SUMMARY
Using a sensitive and specific method for measuring albumin in the urine of three groups consisting of early diabetics, " borderline diabetics ", and normal people, a gradation in the rate of urinary-albumin excretion was found. Diabetics excreted the most and normals the least albumin of the three groups. This gradation in the rate of albumin excretion occurred at concentrations undetectable by ordinary clinical tests. The increased albuminuria may be related to the glomerular basement-membrane changes reported by other workers, but further correlative studies are
required. This work was supported by a research grant from the British Diabetic Association and by a U.S. Public Health Service Grant. Acknowledgments are also due to Mr. N. Veall for preparing the 1251-labelled albumin, and to Mr. M. Field, Miss A. Southon, and Miss J. Williams for technical assistance.
HARRY KEEN Lond, M.R.C.P. C. CHLOUVERAKIS M.B.
8.
Keen, H., Chlouverakis, C. Lancet, 1963, ii, 913.
Department of Medicine Guy’s Hospital, London, S.E.1
M.D.
Athens