Departments of Medicine and Haematology Royal Perth Hospital, Perth
GLYCOSYLATED HEMOGLOBIN CONCENTRATIONS IN PATIENTS WITH DIABETIC NEUROPATHY VINCENT J. MCCANN
RICHARDE. DAVIS
Controversy has existed on the relationship between the degree of diabetic control and the development of diabetic neuropathy. Some authors have found that clinical neuropathy H and impaired nerve conduction s were more prevalent in patients with poorly controlled disease. Others, however, found that although neuropathy was, in general, more common in those with more severe diabetes, no significant correlation could be obtained between estimated control of diabetes and the presence of neuropathy so. They concluded that inadequate control of blood sugar was not the sole determinant in the pathogenesis of polyneuropathy. The occurrence of neuropathy during good control and its occasional precipitation following the institution of control by diet, insulin or oral hypoglycemic agents has been observed 3 and this weakens the case for neuropathy being the result of poor control. One of the main difficulties in resolving this issue has been in defining the degree of diabetic control. This has, in the past, relied upon estimations of blood and urine glucose which are prone to large variations during the day. Recent investigations have indicated that estimations of the concentration of glycosylated hemoglobin (Hb) gives a more reliable indication of the degree of diabetic control 4'~, as it reflects the degree of glucose elevation over a period of several weeks duration. No close correlation was noted in several previous studies between glycosylated Hb concentrations and the presence of microvascular disease 7,9,u However, recent investigations at this center have shown that, in unselected groups of diabetic patients, glycosylated Hb concentrations were significantly higher in insulin-dependent patients with retinopathy than in insulin-dependent patients without retinopathy ~'2 The present investigation compares glycosylated Hb concentrations in patients with neuropathy to those of a carefully matched group of patients without evidence of neuropathy, Key-words: Diabetic neuropathy; GIycosylated hemoglobin. Received: November 18, 1978. Acta diabet. Iat. I6, 205, 1979.
205
GLYCOSYLATED HEMOGLOBIN AND DIABETIC NEUROPATHY
METHODS Glycosylated Hb was estimated in 50 patients with diabetes mellitus and significant neuropathy. Forty-five patients had a symmetrical, predominantly sensory neuropathy defined as loss of pain or light touch sensation or both distally in the feet or lower legs. Eight of these patients had neuropathic ulceration. Two patients had proximal muscle weakness, pain and wasting in the quadriceps with absent knee jerks. The remaining 3 patients had an autonomic neuropathy with two or more of the usual features of this condition, postural hypotension, loss of sinus arrhythmia, or gastrointestinal dysfunction. However, there was some overlap between these groups and 2 of the patients with sensory neuropathy exhibited features of autonomic disturbance. The diagnosis was confirmed by nerve conduction studies in 9 patients, the remainder being accepted on clinical grounds. The group comprised 24 males and 26 females with a mean age of 58.1 '-+- 13.5 years (range 16 to 80 years). Thirty-six patients were receiving insulin, 4 were controlled on diet alone, and 10 were treated with oral hypoglycemic agents. The diabetic control group consisted of randomly selected diabetic outpatients matched for age ( ~ 5 years), sex and therapy, but with no evidence of neuropathy, i.e. no neurological deficit on clinical examination. In particular, there was no loss of pain or light touch sensation, tendon reflexes were normal and no features of autonomic neuropathy were present. The mean age of this group was 57 --+ 13.9 years which is not significantly different from the neuropathy group. The 'normal' .control group consisted of 56 healthy volunteer subjects. Glycosylated Hb was separated by ion exchange chromatography using minicolumns made from 20-ml syringe barrels charged with Biorex 70 cation exchange resin, 200-400 mesh (Bio-Rad Laboratories). Blood samples were anticoagulated with heparin and 0.5 ml of whole blood was washed once with 10 mt of isotonic saline. The saline was removed and 0.8 ml of hemolysing reagent added (0.1 g white saponin, 0.5 g potassium cyanide dissolved in 100 ml distilled water). The mixture was left for 1 rain after which 9.0 ml of buffer was added (5.18 g NaH2PO4.2H20, 1.18 g Na2HPO4 and 0.65 g potassium cyanide dissolved in 1 1 of water, pH 6.7). After vigorous mixing the mixture was centrifuged at 3,000 rpm for 5 min. The clear Hb solution was transferred to the top of the column and allowed to drain into the resin. Gtycosylated Hb was eluted into a flask using 100 ml of the above buffer. The remainder of the Hb was eluted into a second flask with 100 mt of a second buffer (16.22 g Na2HPO4.2H20 and 6.52 g Na2HPO4 dissolved in 1 t of distilled water, pH 6.4). The optical density of the eluates was measured at 413 nm and the percentage of gIycosylated Hb calculated. Duplicate measurements were made on all samples. The method was basically that described by KYNOCH and LEI-IMANN*.
RESULTS The duration of diabetes and glycosylated Hb concentrations in diabetic patients with neuropathy, diabetic patients without neuropathy and control subjects are shown in tab. 1. The mean duration of diabetes in those with neuropathy was 8.9-----7.7 years which is not significantly different from that in those without neuropathy (11.4 ± 8.6 years). 206
v. J, MCCANN~ R. E, DAVIS
groups
duration of diabetes (years)
diabetics w i t h neuropathy diabetics without neuropathy
gtycosylated Hb
(%)
8.9 ± 7.7
13.9 ± 2.4
11.4 ± 8.6
13.6 4- 2.2
control subiects
7.8 4- 0,9
Tab. 1 - Duration of diabetes and glycosylatedHb concentrations in diabetic patients with and without neuropathy and in control subjects. Data are expressed as mean +- SD.
level of glycosylated H b (%)
groups t6.0
diabetics with neuropathy
l0
15
15
10
diabetics without neuropathy
12
15
15
8
Tab. 2 - D i s t r i b u t i o n of g l y c o s y l a t e d H b c o n c e n t r a t i o n s in d i a b e t i c p a t i e n t s w i t h a n d w i t h o u t neuropathy.
In 17 patients neuropathy occurred within 2 years of the onset of diabetes while in the remainder the duration of the diabetes ranged from 2 to more than 30 years, Similarly the mean glycosylated Hb concentration was not significantly different in the two diabetic groups being 13.9% ~ 2.4 (SD) in those with neuropathy and 13.6% ± 2.2 in those without this complication. Both groups of diabetics had a significantly higher mean glycosylated Hb concentration than the 'normal' control group (7.8% ~ 0.9). Table 2 shows the distribution of glycosylated Hb concentrations in both groups of diabetic patients. The number occurring in each range is not significantly different in the neuropathy group compared with those without neuropathy. In fact neuropathy was often present in patients with well controlled diabetes as defined by the degree of hyperglycemia and glycosuria or by the glycosylated Ht~ concentrations. In one patient, for example, an acute sensory neuropathy occurred despite good control of diabetes indicated by a glycosylated Hb level of 8.8% which is within the range of non-diabetics, a blood sugar of 11.6 retool/1 and minimal glycosuria. Similarly a number of patients with poorly controlled diabetes had no evidence of neuropathy on clinical examination.
DISCUSSION In the present investigation there appeared to be no correlation between the development of neuropathy and the degree of control as judged by glycosylated Hb levels or by the more usual criteria of blood sugar estimations and degree of glycosuria. The method of the present investigation does not exclude the possibility of episodes of poor control occurring in the past in those patients with neuropathy. Ideatlv glycosylated Hb levels should be monitored from the onset of diabetes. 207
GLYCOSYLATED HEMOGLOBIN AND DIABETIC N E U R O P A T ~
The results do, however, suggest that those with neuropathy are not significantly less well controlled than those without as it is unlikely that the mean levels of glycosylated Hb would be so similar in the two groups if those with neuropathy were less well controlled. Some of the patients in the neuropathy group had relatively mild diabetes, in four instances controlled by diet alone and the severity and duration of diabetes in the group was not different from that in those without neuropathy. Neuropathy occurred in patients whose diabetes was well controlled and was frequently present early in the course of the disease. It was equally evident that many patients with severe diabetes of long duration had no evidence of neurological deficit on clinical examination and this supports the hypothesis that neuropathy cannot be attributable to hyperglycemia done. The occurrence of neuropathy in mild or well controlled diabetes and its lack of correlation with the duration and severity of the diabetes has been reported previously 3 This has been confirmed by the present study and suggests that 'unlike diabetic retinopathy which is associated with a raised glycosylated Hb level', factors other than the degree or duration of hyperglycemia must exist and may include other metabolic disturbances and possibly genetic factors.
SUMMARY Glycosylated hemoglobin (Hb) concentrations were measured in 50 patients with clinically significant diabetic neuropathy. There were 24 males and 26 females with a mean age of 58.1 years and a mean duration of diabetes of 8.9 years. The glycosylated Hb concentration was not significantly different in these patients (13.9"%--+2.4 SD) compared with randomly selected diabetic patients matched for age ( ± 5 years), sex and therapy without clinical evidence of neuropathy (13.6% -+ 2.2). There was no significant difference in the duration of diabetes between the two groups. The results would suggest that factors other than the degree of control of diabetes are important in the pathogenesis of diabetic neuropathy.
REFERENCES 1) DAvis R.E., CONSTABLE I., NICOL D.J'., McCANN V.J., WELBORN T.: Retinopathy and glycosylated haemoglobin levels in patients with diabetes mellitus - (In press). 2) DAvis R. E., NICOL D. J., McCa,vN V.J., CALDERJ.S.: Glycosylated haemoglobin in patients with diabetes mellitus - Med. J. Aust. 1, 530, 1978. 3) ELLENEE~a M.: Diabetic neuropathy: a consideration of factors in onset - Ann. intern. Med. 52, 1067, 1960. 4) GAEBAY K.H., HASTY K., BRESLOW J.L., ELLISON C.R., BUNN F.H., GALLOP P.M.: Glycosytated hemoglobin and long term blood glucose control in diabetics - J. clin. Endocr. 44, 859, 1977. 5) GRE~XSEN G.: Diabetic neuropathy: influence of age, sex and metabolic control, and duration of diabetes on motor conduction velocity - Neurology (Minneap.) I7, 192, I967. 6) KOENIa R.J., PETERSON C.M., JONES R.L., SAUNDER C., LEHRMAN M., CERA,X~IA.: Correlation of glucose regulation and hemoglobin A l c in diabetes metlitus - New Engl. J. Med. 29Y, 417, 1976. 7) KOENIa R.J., PETERSON C.M., KILO C., CERAM~ A., WILL~AMSON J.R.: Hemoglobin A~ as an indicator of the degree of glucose intolerance in diabetes - Diabetes 25, 230. 1976. 8) KYNOCH P.A.M., LEHMANNH.: Rapid estimation (2~.~ hours) of glycosyIated haemoglobin for routine purposes - Lancet 2, 16, 1977. 9) ~L~LONE J.I., S~MONS C.A., VaN CADE~ T.C.: Correlation of HbAt and diabetic microvascular disease (MVD) - Diabetes 27 (Suppl. 2), 434, 1978; abstract # 15.
208
V,J, MCCANN, R.E. DAVIS 10) L~V~ULDERD. W., LAMBERTE. H., BASTRONJ.A., SPRAGUE R.G.: The neuropathies associated
with diabetes metlitus - Neurology (Minneap.) 1I, 275, 1961. 11) PtRAT J'.: Diabetic neuropathy: a metabolic or vascular disease - Diabetes 14, 1, 1965. 12) TRIVELLI L. A., RANNEYH. M,, Lat H.-T.: Hemoglobin components in patients with diabetes melIitus - New EngL J. Med. 284, 353, 1971.
Requests/or reprints should be addressed to: RICHARD E. DAvIs Department o/ Haematology, Royal Perth Hospital Perth, Western Australia - Aastralia
209
GLYCOSYLATED HEMOGLOBIN CONCENTRATIONS IN PATIENTS WITH DIABETIC NEUROPATHY VINCENT J. MCCANN
RICHARDE. DAVIS
Controversy has existed on the relationship between the degree of diabetic control and the development of diabetic neuropathy. Some authors have found that clinical neuropathy H and impaired nerve conduction s were more prevalent in patients with poorly controlled disease. Others, however, found that although neuropathy was, in general, more common in those with more severe diabetes, no significant correlation could be obtained between estimated control of diabetes and the presence of neuropathy so. They concluded that inadequate control of blood sugar was not the sole determinant in the pathogenesis of polyneuropathy. The occurrence of neuropathy during good control and its occasional precipitation following the institution of control by diet, insulin or oral hypoglycemic agents has been observed 3 and this weakens the case for neuropathy being the result of poor control. One of the main difficulties in resolving this issue has been in defining the degree of diabetic control. This has, in the past, relied upon estimations of blood and urine glucose which are prone to large variations during the day. Recent investigations have indicated that estimations of the concentration of glycosylated hemoglobin (Hb) gives a more reliable indication of the degree of diabetic control 4'~, as it reflects the degree of glucose elevation over a period of several weeks duration. No close correlation was noted in several previous studies between glycosylated Hb concentrations and the presence of microvascular disease 7,9,u However, recent investigations at this center have shown that, in unselected groups of diabetic patients, glycosylated Hb concentrations were significantly higher in insulin-dependent patients with retinopathy than in insulin-dependent patients without retinopathy ~'2 The present investigation compares glycosylated Hb concentrations in patients with neuropathy to those of a carefully matched group of patients without evidence of neuropathy, Key-words: Diabetic neuropathy; GIycosylated hemoglobin. Received: November 18, 1978. Acta diabet. Iat. I6, 205, 1979.
205
GLYCOSYLATED HEMOGLOBIN AND DIABETIC NEUROPATHY
METHODS Glycosylated Hb was estimated in 50 patients with diabetes mellitus and significant neuropathy. Forty-five patients had a symmetrical, predominantly sensory neuropathy defined as loss of pain or light touch sensation or both distally in the feet or lower legs. Eight of these patients had neuropathic ulceration. Two patients had proximal muscle weakness, pain and wasting in the quadriceps with absent knee jerks. The remaining 3 patients had an autonomic neuropathy with two or more of the usual features of this condition, postural hypotension, loss of sinus arrhythmia, or gastrointestinal dysfunction. However, there was some overlap between these groups and 2 of the patients with sensory neuropathy exhibited features of autonomic disturbance. The diagnosis was confirmed by nerve conduction studies in 9 patients, the remainder being accepted on clinical grounds. The group comprised 24 males and 26 females with a mean age of 58.1 '-+- 13.5 years (range 16 to 80 years). Thirty-six patients were receiving insulin, 4 were controlled on diet alone, and 10 were treated with oral hypoglycemic agents. The diabetic control group consisted of randomly selected diabetic outpatients matched for age ( ~ 5 years), sex and therapy, but with no evidence of neuropathy, i.e. no neurological deficit on clinical examination. In particular, there was no loss of pain or light touch sensation, tendon reflexes were normal and no features of autonomic neuropathy were present. The mean age of this group was 57 --+ 13.9 years which is not significantly different from the neuropathy group. The 'normal' .control group consisted of 56 healthy volunteer subjects. Glycosylated Hb was separated by ion exchange chromatography using minicolumns made from 20-ml syringe barrels charged with Biorex 70 cation exchange resin, 200-400 mesh (Bio-Rad Laboratories). Blood samples were anticoagulated with heparin and 0.5 ml of whole blood was washed once with 10 mt of isotonic saline. The saline was removed and 0.8 ml of hemolysing reagent added (0.1 g white saponin, 0.5 g potassium cyanide dissolved in 100 ml distilled water). The mixture was left for 1 rain after which 9.0 ml of buffer was added (5.18 g NaH2PO4.2H20, 1.18 g Na2HPO4 and 0.65 g potassium cyanide dissolved in 1 1 of water, pH 6.7). After vigorous mixing the mixture was centrifuged at 3,000 rpm for 5 min. The clear Hb solution was transferred to the top of the column and allowed to drain into the resin. Gtycosylated Hb was eluted into a flask using 100 ml of the above buffer. The remainder of the Hb was eluted into a second flask with 100 mt of a second buffer (16.22 g Na2HPO4.2H20 and 6.52 g Na2HPO4 dissolved in 1 t of distilled water, pH 6.4). The optical density of the eluates was measured at 413 nm and the percentage of gIycosylated Hb calculated. Duplicate measurements were made on all samples. The method was basically that described by KYNOCH and LEI-IMANN*.
RESULTS The duration of diabetes and glycosylated Hb concentrations in diabetic patients with neuropathy, diabetic patients without neuropathy and control subjects are shown in tab. 1. The mean duration of diabetes in those with neuropathy was 8.9-----7.7 years which is not significantly different from that in those without neuropathy (11.4 ± 8.6 years). 206
v. J, MCCANN~ R. E, DAVIS
groups
duration of diabetes (years)
diabetics w i t h neuropathy diabetics without neuropathy
gtycosylated Hb
(%)
8.9 ± 7.7
13.9 ± 2.4
11.4 ± 8.6
13.6 4- 2.2
control subiects
7.8 4- 0,9
Tab. 1 - Duration of diabetes and glycosylatedHb concentrations in diabetic patients with and without neuropathy and in control subjects. Data are expressed as mean +- SD.
level of glycosylated H b (%)
groups t6.0
diabetics with neuropathy
l0
15
15
10
diabetics without neuropathy
12
15
15
8
Tab. 2 - D i s t r i b u t i o n of g l y c o s y l a t e d H b c o n c e n t r a t i o n s in d i a b e t i c p a t i e n t s w i t h a n d w i t h o u t neuropathy.
In 17 patients neuropathy occurred within 2 years of the onset of diabetes while in the remainder the duration of the diabetes ranged from 2 to more than 30 years, Similarly the mean glycosylated Hb concentration was not significantly different in the two diabetic groups being 13.9% ~ 2.4 (SD) in those with neuropathy and 13.6% ± 2.2 in those without this complication. Both groups of diabetics had a significantly higher mean glycosylated Hb concentration than the 'normal' control group (7.8% ~ 0.9). Table 2 shows the distribution of glycosylated Hb concentrations in both groups of diabetic patients. The number occurring in each range is not significantly different in the neuropathy group compared with those without neuropathy. In fact neuropathy was often present in patients with well controlled diabetes as defined by the degree of hyperglycemia and glycosuria or by the glycosylated Ht~ concentrations. In one patient, for example, an acute sensory neuropathy occurred despite good control of diabetes indicated by a glycosylated Hb level of 8.8% which is within the range of non-diabetics, a blood sugar of 11.6 retool/1 and minimal glycosuria. Similarly a number of patients with poorly controlled diabetes had no evidence of neuropathy on clinical examination.
DISCUSSION In the present investigation there appeared to be no correlation between the development of neuropathy and the degree of control as judged by glycosylated Hb levels or by the more usual criteria of blood sugar estimations and degree of glycosuria. The method of the present investigation does not exclude the possibility of episodes of poor control occurring in the past in those patients with neuropathy. Ideatlv glycosylated Hb levels should be monitored from the onset of diabetes. 207
GLYCOSYLATED HEMOGLOBIN AND DIABETIC N E U R O P A T ~
The results do, however, suggest that those with neuropathy are not significantly less well controlled than those without as it is unlikely that the mean levels of glycosylated Hb would be so similar in the two groups if those with neuropathy were less well controlled. Some of the patients in the neuropathy group had relatively mild diabetes, in four instances controlled by diet alone and the severity and duration of diabetes in the group was not different from that in those without neuropathy. Neuropathy occurred in patients whose diabetes was well controlled and was frequently present early in the course of the disease. It was equally evident that many patients with severe diabetes of long duration had no evidence of neurological deficit on clinical examination and this supports the hypothesis that neuropathy cannot be attributable to hyperglycemia done. The occurrence of neuropathy in mild or well controlled diabetes and its lack of correlation with the duration and severity of the diabetes has been reported previously 3 This has been confirmed by the present study and suggests that 'unlike diabetic retinopathy which is associated with a raised glycosylated Hb level', factors other than the degree or duration of hyperglycemia must exist and may include other metabolic disturbances and possibly genetic factors.
SUMMARY Glycosylated hemoglobin (Hb) concentrations were measured in 50 patients with clinically significant diabetic neuropathy. There were 24 males and 26 females with a mean age of 58.1 years and a mean duration of diabetes of 8.9 years. The glycosylated Hb concentration was not significantly different in these patients (13.9"%--+2.4 SD) compared with randomly selected diabetic patients matched for age ( ± 5 years), sex and therapy without clinical evidence of neuropathy (13.6% -+ 2.2). There was no significant difference in the duration of diabetes between the two groups. The results would suggest that factors other than the degree of control of diabetes are important in the pathogenesis of diabetic neuropathy.
REFERENCES 1) DAvis R.E., CONSTABLE I., NICOL D.J'., McCANN V.J., WELBORN T.: Retinopathy and glycosylated haemoglobin levels in patients with diabetes mellitus - (In press). 2) DAvis R. E., NICOL D. J., McCa,vN V.J., CALDERJ.S.: Glycosylated haemoglobin in patients with diabetes mellitus - Med. J. Aust. 1, 530, 1978. 3) ELLENEE~a M.: Diabetic neuropathy: a consideration of factors in onset - Ann. intern. Med. 52, 1067, 1960. 4) GAEBAY K.H., HASTY K., BRESLOW J.L., ELLISON C.R., BUNN F.H., GALLOP P.M.: Glycosytated hemoglobin and long term blood glucose control in diabetics - J. clin. Endocr. 44, 859, 1977. 5) GRE~XSEN G.: Diabetic neuropathy: influence of age, sex and metabolic control, and duration of diabetes on motor conduction velocity - Neurology (Minneap.) I7, 192, I967. 6) KOENIa R.J., PETERSON C.M., JONES R.L., SAUNDER C., LEHRMAN M., CERA,X~IA.: Correlation of glucose regulation and hemoglobin A l c in diabetes metlitus - New Engl. J. Med. 29Y, 417, 1976. 7) KOENIa R.J., PETERSON C.M., KILO C., CERAM~ A., WILL~AMSON J.R.: Hemoglobin A~ as an indicator of the degree of glucose intolerance in diabetes - Diabetes 25, 230. 1976. 8) KYNOCH P.A.M., LEHMANNH.: Rapid estimation (2~.~ hours) of glycosyIated haemoglobin for routine purposes - Lancet 2, 16, 1977. 9) ~L~LONE J.I., S~MONS C.A., VaN CADE~ T.C.: Correlation of HbAt and diabetic microvascular disease (MVD) - Diabetes 27 (Suppl. 2), 434, 1978; abstract # 15.
208
V,J, MCCANN, R.E. DAVIS 10) L~V~ULDERD. W., LAMBERTE. H., BASTRONJ.A., SPRAGUE R.G.: The neuropathies associated
with diabetes metlitus - Neurology (Minneap.) 1I, 275, 1961. 11) PtRAT J'.: Diabetic neuropathy: a metabolic or vascular disease - Diabetes 14, 1, 1965. 12) TRIVELLI L. A., RANNEYH. M,, Lat H.-T.: Hemoglobin components in patients with diabetes melIitus - New EngL J. Med. 284, 353, 1971.
Requests/or reprints should be addressed to: RICHARD E. DAvIs Department o/ Haematology, Royal Perth Hospital Perth, Western Australia - Aastralia
209
