J o u r ~ a of l lnturial Medicine 1990: 2 2 7 : 133-1 36
Glycosylated haemoglobin (HbA,,) in iron- and vitamin B12 deficiency 1’. GRAM-HANSEN, J. EKIKSEN, 1’. MOURITS-ANDEKSEN 81 I,. OLESEN From the llepartrnetit oj’ lriternnl Medicine H. Sectiori of Herncitologg, Anlliorg Hospitcil. Acrlborg. Ilerirriork
Abstract. Gram-Hansen P, Eriksen J, Mouritis-Andersen T, Olesen L (Department of Internal Medicine B. Section of Hematology. Aalborg Hospital, Aalborg. Ilenmark). Glycosylated haemoglobin (HbA,,J in iron and vitamin I31 2 deficiency. lourrial of Internal Medcine 1990: 227: 133-136. Glycosylated haemoglobin (HbA,,) was measured in 10 patients with iron deficiency anaemia, 10 patients with vitamin B12 deficiency anaemia and 10 healthy controls. Initially there were no significant differences between the groups ( P > 0.4). but after treatment with iron and vitamin B12 for 3 and 6 weeks, the glycosylated haemoglobin concentration decreased significantly (P < 0.01). It was concluded that glycosylated haemoglobin is a sensitive marker of the changes in the erythrocyte population that are observed when predominantly immature erythrocytes are being produced. Keywords: glycosylated haemoglobin (HbAJ. iron deficiency anaemia, vitamin B12 deficiency anaemia.
Introduction
Methods
Glycosylated haemoglobin (HbA,,) is formed by a progressive, non-enzymatic reaction between glucose and haemoglobin within the erythrocytes [l]. The HbA,, concentration is dependent on the plasma glucose level and the stage of development of the erythrocytes, i.e. red cell life span [2]. Young erythrocytes contain lower levels of glycosylated haemoglobin than mature erythrocytes, and HbA,, can therefore be used not only to predict glucose levels in diabetics [ 3 ] , but also as a diagnostic parameter of anaemia with reduced red cell survival rate [4]. In anaemia that is not characterized by an excess of immature erythrocytes, it would be expected that the concentration of HbA,, would remain unchanged. [5-91. This phenomenon has been reported TS-91 in iron and vitamin B12 deficiency anaemia, but the results are conflicting. The aim of the present study was to monitor the HbA,,. concentration in patients with iron and vitamin B12 deficiency anaemia, particularly with regard to changes in HbA,, levels when new, immature erythrocytes are formed in response to treatment.
Ten patients with iron deficiency anaemia, 10 patients with vitamin B12 deficiency anaemia and 10 healthy controls were studied. They were all nondiabetic, and glucose tolerance tests gave normal results. The median age of individuals was 52 years (range 27-81 years) in the group with iron deficiency, 74 years (range 66-82 years) in the group with vitamin B12 deficiency, and 6 0 years (range 50-72 years) in the controls. Glycosylated haemoglobin (HbA,,:) concentration, haemoglobin and erythrocyte parameters were measured 0, 3 , 6 and 9 weeks after initiation of treatment with iron and vitamin 1312. The HbA,, concentration was measured by Fast Protein Liquid Chromatography (FPLC) system (ion-exchange chromatography) [ 101. The reference range was 4.2-5.60/, for HbA,,. All patients gave their informed consent according to the Helsinki Declaration. Statistical treatment of the data was by nonparametric analysis using a Mann-Whitney rank sum test for comparison between the groups and a Wilcoxon rank sum test for analysis of data within a group. The significance level was taken to be P < 0.05. 133
P. GRAM-HANSEN et al.
134
Table 1. Median values for glycosylated haemoglobin (HbA,,) concentration and erythrocyte indices in 10 patients with iron deficiency anaemia, 0 and 3 weeks after iron therapy (range shown in parentheses) Time (weeks) 0 3 Reference range
HbA,, (%)
Haemoglobin (mrnol/l)
MCV
4.9(4.0-5.9) 4.3(3.4-5.4)* 4.2-5.6
5.1(3.7-7.2) 7.2(5.9-8.0)* 7.5-1 0.4
66(50-75) 19(8-50) 79(70-92)* 25(8-50)? 85-100 < 20
~
Rcticulocytes (x
~
109/1)
~
~~
' P < 0.01, t P > 0.1.
Table 2. Median values for glycosylated haemoglobin (HbA,,) concentration and erythrocyte indices in 10 patients with vitamin B3 2 deficiency anaemia, 0 and 3 weeks after therapy with vitamin BIZ (range shown in parentheses) Time (weeks)
0 3 Reference range
HbA,, (%)
Haemoglobin (mmol/l)
5.1(4.3-5.8) 4.4(4.0-5.6)* 4.2-5.6
6.1(4.4-7.5) 8.3(6.2-9.1)* 7.5-10.4
MCV 128(106-159) 110(100-1 15)*
85-100
Reticulocytes ( x 10911)
8(4-20) 15(2-30)* < 20
*P < 0.01
-15.0
2
3 -
D
I
Fig. 1, Glycosylated haemoglobin
2 -
Time (weeks)
(HbA,,) concentration in 1 0 patients with iron deficiency anaemia and 10 healthy controls, on initiation of iron therapy and 3, 6 and 9 weeks thereafter. Median values and range are shown. Controls. V ( n = 1 0 ) . Iron deficiency, 0 ( t i = 1 0 ) . --- = reference range.
The median glycosylated haemoglobin (HbA,,.) concentration was 4.9 % (range 4.0-5.9 %) in the group with iron deficiency, 5.1% (range 4.3-5.80/,) in the group with vitamin B12 deficiency and 5.0% (range 4.6-5.9 %) in the controls. There was no significant difference between the groups (P > 0.4). Tables 1 and 2 show the HbA,, concentration and
erythrocyte indices on initiation of treatment with iron and vitamin B12, and 3 weeks later. Figs 1 and 2 show the HbA,, concentration at time 0 and 3, 6 and 9 weeks after the commencement of therapy. There was a significant difference between the initial HbA,, concentration and the HbA,,: levels 3 and 6 weeks after therapy in both groups (P < 0.01). There was a decrease in HbA,, concentration in all patients, and in the two groups 7 patients (35%) showed a
I
-
0
I 0
L 3
6
Results
9
GLYCOSYLATED HAEMOGLOBIN (HbA,,)
135
6
5
--8
4
an
3
I
Fig. 2. Glycosylated haemoglobin (HbA,,.) concentration in 10 patients with vitamin B12 deticiency anaemia and 10 healthy controls. on initiation of cobolaniin therapy and 3 . 6 and 9 weeks thereafter. Median values and range are shown. Controls, V ( n = 10). Vitamin B12 deficiency. ( r t = 10).--- = reference range.
2
I
I
0-
decrease at 3 weeks to below the lower value in the reference range. All patients exhibited a significant change in haemoglobin and erythrocyte indices, except with regard to the reticulocytes in the group with iron deficiency anaemia.
Discussion Glycosylated haemoglobin (HbA,,) is formed by a progressive, non-enzymatic reaction within the erythrocytes. The concentration of HbA,, reflects not only the plasma glucose level, but also the red blood cell survival rate. In haemolytic anaemia, the erythrocyte life span is much reduced, with extensive destruction of primarily mature erythrocytes, and a compensatory increase in production of new, immature red blood cells. Thus a low HbA,, concentration would be expected, and this has been confirmed in a study of non-diabetic patients with haemolytic anaemia [4]. In iron deficiency anaemia, the erythrocyte survival rate is normal, while in vitamin B12 deficiency the red cell survival rate is decreased. The haemolytic component is often minor, and may affect both mature and immature erythrocytes. Therefore normal levels of glycosylated haemoglobin would be expected. On treatment with iron and vitamin B12, bone marrow erythropoiesis is stimulated, with excess production of new, immature erythrocytes, and the HbA,, concentration would be expected to decrease.
1
Brooks et al. [6] observed a higher HbA,, concentration in patients with iron deficiency, with a significant decrease on treatment with iron. Heyningen et al. [7] investigated a similar group of patients, but found no difference in the HbA,, levels compared to a group of controls, nor were there any changes after iron therapy. With regard to vitamin B12 deficiency, no studies have been undertaken. In the present paper we report a normal HbA,, concentration in patients wth iron and vitamin B12 deficiency. After iron and cobalamin therapy, a significant decrease in HbA,, concentration was seen in both groups, together with significant changes in the erythrocyte indices. It is concluded that, in patients with iron- and vitamin B12 deficiency, glycosylated haemoglobin is an important marker of the changes in the erythrocyte population that occur when therapy is initiated.
References 1 Bunn HF. Haney DN. Kamin S. Gabbay KH. Gallop PM. The biosynthesis of human hemoglobin Ale. Slow glycosylation of hemoglobin in vivo. J Clin Invest 1 9 7 6 : 5 7 : 1652-59. 2 Fitzgibbons JF. Koler RD. Jones RT. Red cell age-related changes of hemoglobins A,,,, and AlCin normal and diabetic subjects. J Clin Invest 1 9 7 6 : 5 8 : 820-24. 3 Ditzel J, KjaergPrd JJ. Haemoglobin A,, concentration after initial treatment for newly discovered diabetes. Br Med J 1 9 7 8 : 1 : 741-2. 4 Panzer S. Kronik G. Lechner K. Bettelheim P. Neumann E. 10-2
136
5
6 7 8
9
P. G R A M - H A N S E N et al.
Dudczak R. Glycosylated hemoglobins (GHb): an index of red cell survival. Blood 1982: 59: 1348-50. Horton BF. Huisman THJ. Studies on the heterogenity of hemoglobin. VII. Minor hemoglobin components in haematological diseases. Br 1 Haematol 1965: 11: 296-304. Brooks AP, Metcalfe I. Day JL. Edwards M. Iron deficiency and haemoglobin A,. Lancet 1980; ii: 141. Heyningen C, Dalton RG. Glycosylated haemoglobin in iron deficiency anaemia. Lancet 1985: 13: l(8433): 332-3. Rai KB. Pattabiraman TN. Glycosylated haemoglobin levels in iron deficiency anaemia. Indian Med Res 1986; 8 3 : 234-6. Davis RE, McCann VJ, Nicol DJ. Influence of iron-deficiency
anaemia on the glycosylated haemoglobin level in a patient with diabetes mellitus. Med 1 Aust 1983: 8 : 1 ( 1 ) : 40-1. 10 Jeppson ]A. Jerntorp P. Sundkvist G. Englund H. Nylund V. Measurement of hemoglobin A,r by a new liquid-chromatographic assay: methodology. clinical utility, and relation to glucose tolerance evaluated. Clin C h e m 1986: 3 2 : 1867-72. Received 29 June 1989, accepted 8 August 1989.
Correspondence: Dr Paul Gram-Hansen. Beatesmindevej 52.2.th.. DK-92 1 0 Aalborg. Denmark.
Glycosylated haemoglobin (HbA,,) in iron- and vitamin B12 deficiency 1’. GRAM-HANSEN, J. EKIKSEN, 1’. MOURITS-ANDEKSEN 81 I,. OLESEN From the llepartrnetit oj’ lriternnl Medicine H. Sectiori of Herncitologg, Anlliorg Hospitcil. Acrlborg. Ilerirriork
Abstract. Gram-Hansen P, Eriksen J, Mouritis-Andersen T, Olesen L (Department of Internal Medicine B. Section of Hematology. Aalborg Hospital, Aalborg. Ilenmark). Glycosylated haemoglobin (HbA,,J in iron and vitamin I31 2 deficiency. lourrial of Internal Medcine 1990: 227: 133-136. Glycosylated haemoglobin (HbA,,) was measured in 10 patients with iron deficiency anaemia, 10 patients with vitamin B12 deficiency anaemia and 10 healthy controls. Initially there were no significant differences between the groups ( P > 0.4). but after treatment with iron and vitamin B12 for 3 and 6 weeks, the glycosylated haemoglobin concentration decreased significantly (P < 0.01). It was concluded that glycosylated haemoglobin is a sensitive marker of the changes in the erythrocyte population that are observed when predominantly immature erythrocytes are being produced. Keywords: glycosylated haemoglobin (HbAJ. iron deficiency anaemia, vitamin B12 deficiency anaemia.
Introduction
Methods
Glycosylated haemoglobin (HbA,,) is formed by a progressive, non-enzymatic reaction between glucose and haemoglobin within the erythrocytes [l]. The HbA,, concentration is dependent on the plasma glucose level and the stage of development of the erythrocytes, i.e. red cell life span [2]. Young erythrocytes contain lower levels of glycosylated haemoglobin than mature erythrocytes, and HbA,, can therefore be used not only to predict glucose levels in diabetics [ 3 ] , but also as a diagnostic parameter of anaemia with reduced red cell survival rate [4]. In anaemia that is not characterized by an excess of immature erythrocytes, it would be expected that the concentration of HbA,, would remain unchanged. [5-91. This phenomenon has been reported TS-91 in iron and vitamin B12 deficiency anaemia, but the results are conflicting. The aim of the present study was to monitor the HbA,,. concentration in patients with iron and vitamin B12 deficiency anaemia, particularly with regard to changes in HbA,, levels when new, immature erythrocytes are formed in response to treatment.
Ten patients with iron deficiency anaemia, 10 patients with vitamin B12 deficiency anaemia and 10 healthy controls were studied. They were all nondiabetic, and glucose tolerance tests gave normal results. The median age of individuals was 52 years (range 27-81 years) in the group with iron deficiency, 74 years (range 66-82 years) in the group with vitamin B12 deficiency, and 6 0 years (range 50-72 years) in the controls. Glycosylated haemoglobin (HbA,,:) concentration, haemoglobin and erythrocyte parameters were measured 0, 3 , 6 and 9 weeks after initiation of treatment with iron and vitamin 1312. The HbA,, concentration was measured by Fast Protein Liquid Chromatography (FPLC) system (ion-exchange chromatography) [ 101. The reference range was 4.2-5.60/, for HbA,,. All patients gave their informed consent according to the Helsinki Declaration. Statistical treatment of the data was by nonparametric analysis using a Mann-Whitney rank sum test for comparison between the groups and a Wilcoxon rank sum test for analysis of data within a group. The significance level was taken to be P < 0.05. 133
P. GRAM-HANSEN et al.
134
Table 1. Median values for glycosylated haemoglobin (HbA,,) concentration and erythrocyte indices in 10 patients with iron deficiency anaemia, 0 and 3 weeks after iron therapy (range shown in parentheses) Time (weeks) 0 3 Reference range
HbA,, (%)
Haemoglobin (mrnol/l)
MCV
4.9(4.0-5.9) 4.3(3.4-5.4)* 4.2-5.6
5.1(3.7-7.2) 7.2(5.9-8.0)* 7.5-1 0.4
66(50-75) 19(8-50) 79(70-92)* 25(8-50)? 85-100 < 20
~
Rcticulocytes (x
~
109/1)
~
~~
' P < 0.01, t P > 0.1.
Table 2. Median values for glycosylated haemoglobin (HbA,,) concentration and erythrocyte indices in 10 patients with vitamin B3 2 deficiency anaemia, 0 and 3 weeks after therapy with vitamin BIZ (range shown in parentheses) Time (weeks)
0 3 Reference range
HbA,, (%)
Haemoglobin (mmol/l)
5.1(4.3-5.8) 4.4(4.0-5.6)* 4.2-5.6
6.1(4.4-7.5) 8.3(6.2-9.1)* 7.5-10.4
MCV 128(106-159) 110(100-1 15)*
85-100
Reticulocytes ( x 10911)
8(4-20) 15(2-30)* < 20
*P < 0.01
-15.0
2
3 -
D
I
Fig. 1, Glycosylated haemoglobin
2 -
Time (weeks)
(HbA,,) concentration in 1 0 patients with iron deficiency anaemia and 10 healthy controls, on initiation of iron therapy and 3, 6 and 9 weeks thereafter. Median values and range are shown. Controls. V ( n = 1 0 ) . Iron deficiency, 0 ( t i = 1 0 ) . --- = reference range.
The median glycosylated haemoglobin (HbA,,.) concentration was 4.9 % (range 4.0-5.9 %) in the group with iron deficiency, 5.1% (range 4.3-5.80/,) in the group with vitamin B12 deficiency and 5.0% (range 4.6-5.9 %) in the controls. There was no significant difference between the groups (P > 0.4). Tables 1 and 2 show the HbA,, concentration and
erythrocyte indices on initiation of treatment with iron and vitamin B12, and 3 weeks later. Figs 1 and 2 show the HbA,, concentration at time 0 and 3, 6 and 9 weeks after the commencement of therapy. There was a significant difference between the initial HbA,, concentration and the HbA,,: levels 3 and 6 weeks after therapy in both groups (P < 0.01). There was a decrease in HbA,, concentration in all patients, and in the two groups 7 patients (35%) showed a
I
-
0
I 0
L 3
6
Results
9
GLYCOSYLATED HAEMOGLOBIN (HbA,,)
135
6
5
--8
4
an
3
I
Fig. 2. Glycosylated haemoglobin (HbA,,.) concentration in 10 patients with vitamin B12 deticiency anaemia and 10 healthy controls. on initiation of cobolaniin therapy and 3 . 6 and 9 weeks thereafter. Median values and range are shown. Controls, V ( n = 10). Vitamin B12 deficiency. ( r t = 10).--- = reference range.
2
I
I
0-
decrease at 3 weeks to below the lower value in the reference range. All patients exhibited a significant change in haemoglobin and erythrocyte indices, except with regard to the reticulocytes in the group with iron deficiency anaemia.
Discussion Glycosylated haemoglobin (HbA,,) is formed by a progressive, non-enzymatic reaction within the erythrocytes. The concentration of HbA,, reflects not only the plasma glucose level, but also the red blood cell survival rate. In haemolytic anaemia, the erythrocyte life span is much reduced, with extensive destruction of primarily mature erythrocytes, and a compensatory increase in production of new, immature red blood cells. Thus a low HbA,, concentration would be expected, and this has been confirmed in a study of non-diabetic patients with haemolytic anaemia [4]. In iron deficiency anaemia, the erythrocyte survival rate is normal, while in vitamin B12 deficiency the red cell survival rate is decreased. The haemolytic component is often minor, and may affect both mature and immature erythrocytes. Therefore normal levels of glycosylated haemoglobin would be expected. On treatment with iron and vitamin B12, bone marrow erythropoiesis is stimulated, with excess production of new, immature erythrocytes, and the HbA,, concentration would be expected to decrease.
1
Brooks et al. [6] observed a higher HbA,, concentration in patients with iron deficiency, with a significant decrease on treatment with iron. Heyningen et al. [7] investigated a similar group of patients, but found no difference in the HbA,, levels compared to a group of controls, nor were there any changes after iron therapy. With regard to vitamin B12 deficiency, no studies have been undertaken. In the present paper we report a normal HbA,, concentration in patients wth iron and vitamin B12 deficiency. After iron and cobalamin therapy, a significant decrease in HbA,, concentration was seen in both groups, together with significant changes in the erythrocyte indices. It is concluded that, in patients with iron- and vitamin B12 deficiency, glycosylated haemoglobin is an important marker of the changes in the erythrocyte population that occur when therapy is initiated.
References 1 Bunn HF. Haney DN. Kamin S. Gabbay KH. Gallop PM. The biosynthesis of human hemoglobin Ale. Slow glycosylation of hemoglobin in vivo. J Clin Invest 1 9 7 6 : 5 7 : 1652-59. 2 Fitzgibbons JF. Koler RD. Jones RT. Red cell age-related changes of hemoglobins A,,,, and AlCin normal and diabetic subjects. J Clin Invest 1 9 7 6 : 5 8 : 820-24. 3 Ditzel J, KjaergPrd JJ. Haemoglobin A,, concentration after initial treatment for newly discovered diabetes. Br Med J 1 9 7 8 : 1 : 741-2. 4 Panzer S. Kronik G. Lechner K. Bettelheim P. Neumann E. 10-2
136
5
6 7 8
9
P. G R A M - H A N S E N et al.
Dudczak R. Glycosylated hemoglobins (GHb): an index of red cell survival. Blood 1982: 59: 1348-50. Horton BF. Huisman THJ. Studies on the heterogenity of hemoglobin. VII. Minor hemoglobin components in haematological diseases. Br 1 Haematol 1965: 11: 296-304. Brooks AP, Metcalfe I. Day JL. Edwards M. Iron deficiency and haemoglobin A,. Lancet 1980; ii: 141. Heyningen C, Dalton RG. Glycosylated haemoglobin in iron deficiency anaemia. Lancet 1985: 13: l(8433): 332-3. Rai KB. Pattabiraman TN. Glycosylated haemoglobin levels in iron deficiency anaemia. Indian Med Res 1986; 8 3 : 234-6. Davis RE, McCann VJ, Nicol DJ. Influence of iron-deficiency
anaemia on the glycosylated haemoglobin level in a patient with diabetes mellitus. Med 1 Aust 1983: 8 : 1 ( 1 ) : 40-1. 10 Jeppson ]A. Jerntorp P. Sundkvist G. Englund H. Nylund V. Measurement of hemoglobin A,r by a new liquid-chromatographic assay: methodology. clinical utility, and relation to glucose tolerance evaluated. Clin C h e m 1986: 3 2 : 1867-72. Received 29 June 1989, accepted 8 August 1989.
Correspondence: Dr Paul Gram-Hansen. Beatesmindevej 52.2.th.. DK-92 1 0 Aalborg. Denmark.
