- Email: [email protected]
PROTEIN BROWNING AND DIABETIC COMPLICATIONS
459
for up to 12°o of all significant mycobacterial isolates from extrapulmonary sites.6 Because M bovis lacks the amidase enzyme necessary for converting pyrazinamide into pyrazinoic acid, with its lethal effect, an alternative drug should be chosen. Another important consideration in the United Kingdom is the extent to which the size of the immigrant community can influence the frequency and pattern of non-respiratory disease and the isolation rate of drug-resistant strains. In Liverpool, for instance, where the proportion , of the population who are New Commonwealth immigrants is one quarter of the national average,6 genitourinary tuberculosis is twice as common as tuberculous lymphadenitis’ (a reversal of the national figures)8 and drug resistance is rare (unpublished). This reveals the association between Europeans and genitourinary disease, and Asians and lymphadenitis.9 Finally, no review of the management of non-respiratory tuberculosis is complete without mention of the role of surgery in removing sizeable collections of caseous material (eg, psoas or paravertebral abscesses) or in relieving mechanical problems (eg, pericardiectomy in constrictive pericarditis). account
Public Health Laboratory Service, Fazakerley Hospital, Liverpool L9 7AL
E. G. L. WILKINS C. ROBERTS
1 Mitchison DA. The action of antituberculosis
drugs m short-course chemotherapy. Tubercle 1985; 66: 219-25. 2 Girling DJ The role of pyrazinamide in primary chemotherapy for pulmonary tuberculosis. Tubercle 1984; 65: 1-4. 3. East and Central African/British Medical Research Council fifth collaborative study. Controlled clinical trial of 4 short-course regimens of chemotherapy (three 6-month and one 8-month) for pulmonary tuberculosis: final report. Tubercle 1986; 67: 5-15. 4. Ross JD, Home NW. Modem drug treatment in tuberculosis. London: Chest, Heart and Stroke Association, 1983: 60-64. 5. Forgan-Smith R, Ellard GA, Newton D, Mitchison DA. Pyrazinamide and other drugs in tuberculous meningitis. Lancet 1973; ii: 374 6. Wilkins EGL, Griffiths RJ, Roberts C. Bovine variants of Mycobacterium tuberculosis isolated in Liverpool during the period 1969 to 1983: an epidemiological survey. 1986; 59: 627-35. Quart Med J 7. Census 1981, Great Britain. National and regional summary. London: HMSO, 1983. 8. Medical Research Council Tuberculosis and Chest Diseases Unit. National survey of tuberculosis notifications in England and Wales. Br Med J 1980; 281: 895-98. 9. Collins CH, Yates MD, Grange JM. A study of bovine strains of Mycobacterium tuberculosis isolated from humans in South East England, 1977-1979. Tubercle 1981; 62: 113-16
Effect of free radicals
aggregation and fluorescence of IgG and
IgG glycosylated in vitro and a control sample of protein were separated by HPLC2 before and after 15 min of ultraviolet irradiation which induces the formation of free radicals. absorbance emission 454 nm),
-
PROTEIN BROWNING AND DIABETIC
on
glycated IgG.
=
at
280 nm;
= .....
fluorescence (excitation 360 nm,
COMPLICATIONS
SIR,—Your May 24 editorial on non-enzymatic protein glycosylation, browning, and diabetic microangiopathy emphasised the need for further biochemical investigation of such browning reactions. We present here some further evidence and an alternative interpretation of the mechanism. Non-enzymatically glycosylated proteins which have undergone browning reactions are fluorescent (excitation 370 nm, emission 440 nm), and such fluorescence has been described in those diabetic proteins, such as collagen,l that have a long half-life. The use of a sensitive assay2 has allowed us to detect similar fluorescence (excitation 360 nm, emission 454 nm) in the albumin and globulin fractions from diabetic sera. An association was found between’the degree of fluorescence of high-molecular-weight serum protein, principally gammaglobulins, and microangiopathy.3 Moreover there was no significant correlation between serum protein fluorescence and the level of blood glucose control, assessed by measurement of HbA1c or fructosamine. Thus protein browning, defined by such fluorescence, appears to be a property of non-enzymatically glycosylated proteins irrespective of their halflife and is not directly proportional to their degree of glycation. We have found2 that protein aggregation and fluorescence (excitation 360 nm, emission 454 nm) can both be caused by free radical mechanisms (see figure). The oxidation of cysteine and aromatic aminoacid residues by free radicals leads to the formation of sulphoxides and highly fluorescent kynurenines.4 Nonenzymatic glycosylation of protein confers an increased susceptibility to this free-radical-mediated fluorescence and aggregation (figure). Although the reason for this effect of glycosylation is unclear, we have demonstrated that protein
ketoamines, the stable adducts of glucose with protein amino can undergo an autoxidation in which at least one free radical, the superoxide anion, is generated. Non-enzymatic glycosylation may enhance protein oxidation and aggregation by virtue of the ability of ketoamine-bound glucose to participate in
groups,
free-radical reactions. Several features of the browning reactions of glycosylated proteins are consistent with a free-radical-mediated process. This. lends further support to the suggestion that free-radical processes may be implicated in the pathogenesis of diabetic microangiopathy.6
Selly Oak Hospital, Birmingham B29 6JD
A. F. JONES P. E. JENNINGS .A. WAKEFIELD J. W. WINKLES J. LUNEC A. H. BARNETT
1. Monnier VM, Vishwanath V, Frank KE, Elmets CA, Dauchot P, Kohn RR. Relation
between complications of type 1 diabetes and collagen-linked fluorescence. N Engl J Med 1986; 314: 403-08. 2. Lunec J, Blake DR, McCleary SJ, Brailsford S, Bacon PA. Self perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation. J Clin Invest 1985; 76: 2084-90. Free radical oxidation of serum proteins in diabetics with and without microangiopathy. Diab Med (in press). 4. Roshchupkin DI, Talitsky VV, Pelenitsyn AB. Fluormerric study of tryptophan photolysis. Photochem Photobiol 1979; 30: 635-43. 5. Jones AF, Winkles JW, Lunec J, Jennings PE, Barnett AH. Superoxide radical generation by glycated protein. Clin Sci (in press). 6. Jennings PE, Jones AF, Lunec J, Barnett AH. Free radical reaction products in diabetics with microangiopathy. Diab Med 1985; 2: 502A.
3.
Jennings PE, Jones AF, Wakefield A, Winkles JW, Lunec J, Barnett AH. mediated
for up to 12°o of all significant mycobacterial isolates from extrapulmonary sites.6 Because M bovis lacks the amidase enzyme necessary for converting pyrazinamide into pyrazinoic acid, with its lethal effect, an alternative drug should be chosen. Another important consideration in the United Kingdom is the extent to which the size of the immigrant community can influence the frequency and pattern of non-respiratory disease and the isolation rate of drug-resistant strains. In Liverpool, for instance, where the proportion , of the population who are New Commonwealth immigrants is one quarter of the national average,6 genitourinary tuberculosis is twice as common as tuberculous lymphadenitis’ (a reversal of the national figures)8 and drug resistance is rare (unpublished). This reveals the association between Europeans and genitourinary disease, and Asians and lymphadenitis.9 Finally, no review of the management of non-respiratory tuberculosis is complete without mention of the role of surgery in removing sizeable collections of caseous material (eg, psoas or paravertebral abscesses) or in relieving mechanical problems (eg, pericardiectomy in constrictive pericarditis). account
Public Health Laboratory Service, Fazakerley Hospital, Liverpool L9 7AL
E. G. L. WILKINS C. ROBERTS
1 Mitchison DA. The action of antituberculosis
drugs m short-course chemotherapy. Tubercle 1985; 66: 219-25. 2 Girling DJ The role of pyrazinamide in primary chemotherapy for pulmonary tuberculosis. Tubercle 1984; 65: 1-4. 3. East and Central African/British Medical Research Council fifth collaborative study. Controlled clinical trial of 4 short-course regimens of chemotherapy (three 6-month and one 8-month) for pulmonary tuberculosis: final report. Tubercle 1986; 67: 5-15. 4. Ross JD, Home NW. Modem drug treatment in tuberculosis. London: Chest, Heart and Stroke Association, 1983: 60-64. 5. Forgan-Smith R, Ellard GA, Newton D, Mitchison DA. Pyrazinamide and other drugs in tuberculous meningitis. Lancet 1973; ii: 374 6. Wilkins EGL, Griffiths RJ, Roberts C. Bovine variants of Mycobacterium tuberculosis isolated in Liverpool during the period 1969 to 1983: an epidemiological survey. 1986; 59: 627-35. Quart Med J 7. Census 1981, Great Britain. National and regional summary. London: HMSO, 1983. 8. Medical Research Council Tuberculosis and Chest Diseases Unit. National survey of tuberculosis notifications in England and Wales. Br Med J 1980; 281: 895-98. 9. Collins CH, Yates MD, Grange JM. A study of bovine strains of Mycobacterium tuberculosis isolated from humans in South East England, 1977-1979. Tubercle 1981; 62: 113-16
Effect of free radicals
aggregation and fluorescence of IgG and
IgG glycosylated in vitro and a control sample of protein were separated by HPLC2 before and after 15 min of ultraviolet irradiation which induces the formation of free radicals. absorbance emission 454 nm),
-
PROTEIN BROWNING AND DIABETIC
on
glycated IgG.
=
at
280 nm;
= .....
fluorescence (excitation 360 nm,
COMPLICATIONS
SIR,—Your May 24 editorial on non-enzymatic protein glycosylation, browning, and diabetic microangiopathy emphasised the need for further biochemical investigation of such browning reactions. We present here some further evidence and an alternative interpretation of the mechanism. Non-enzymatically glycosylated proteins which have undergone browning reactions are fluorescent (excitation 370 nm, emission 440 nm), and such fluorescence has been described in those diabetic proteins, such as collagen,l that have a long half-life. The use of a sensitive assay2 has allowed us to detect similar fluorescence (excitation 360 nm, emission 454 nm) in the albumin and globulin fractions from diabetic sera. An association was found between’the degree of fluorescence of high-molecular-weight serum protein, principally gammaglobulins, and microangiopathy.3 Moreover there was no significant correlation between serum protein fluorescence and the level of blood glucose control, assessed by measurement of HbA1c or fructosamine. Thus protein browning, defined by such fluorescence, appears to be a property of non-enzymatically glycosylated proteins irrespective of their halflife and is not directly proportional to their degree of glycation. We have found2 that protein aggregation and fluorescence (excitation 360 nm, emission 454 nm) can both be caused by free radical mechanisms (see figure). The oxidation of cysteine and aromatic aminoacid residues by free radicals leads to the formation of sulphoxides and highly fluorescent kynurenines.4 Nonenzymatic glycosylation of protein confers an increased susceptibility to this free-radical-mediated fluorescence and aggregation (figure). Although the reason for this effect of glycosylation is unclear, we have demonstrated that protein
ketoamines, the stable adducts of glucose with protein amino can undergo an autoxidation in which at least one free radical, the superoxide anion, is generated. Non-enzymatic glycosylation may enhance protein oxidation and aggregation by virtue of the ability of ketoamine-bound glucose to participate in
groups,
free-radical reactions. Several features of the browning reactions of glycosylated proteins are consistent with a free-radical-mediated process. This. lends further support to the suggestion that free-radical processes may be implicated in the pathogenesis of diabetic microangiopathy.6
Selly Oak Hospital, Birmingham B29 6JD
A. F. JONES P. E. JENNINGS .A. WAKEFIELD J. W. WINKLES J. LUNEC A. H. BARNETT
1. Monnier VM, Vishwanath V, Frank KE, Elmets CA, Dauchot P, Kohn RR. Relation
between complications of type 1 diabetes and collagen-linked fluorescence. N Engl J Med 1986; 314: 403-08. 2. Lunec J, Blake DR, McCleary SJ, Brailsford S, Bacon PA. Self perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation. J Clin Invest 1985; 76: 2084-90. Free radical oxidation of serum proteins in diabetics with and without microangiopathy. Diab Med (in press). 4. Roshchupkin DI, Talitsky VV, Pelenitsyn AB. Fluormerric study of tryptophan photolysis. Photochem Photobiol 1979; 30: 635-43. 5. Jones AF, Winkles JW, Lunec J, Jennings PE, Barnett AH. Superoxide radical generation by glycated protein. Clin Sci (in press). 6. Jennings PE, Jones AF, Lunec J, Barnett AH. Free radical reaction products in diabetics with microangiopathy. Diab Med 1985; 2: 502A.
3.
Jennings PE, Jones AF, Wakefield A, Winkles JW, Lunec J, Barnett AH. mediated