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Clinica Chimica Acta, 93 (1979) 199-205 @ Elsevier/North-Holland Biomedical Press

CGA 10037

CHROMATOGRAPHIC AND COLORIMETRIC DETECTION OF GLYCOSYLATED HEMOGLOBINS: A COMPARATIVE ANALYSIS OF TWO DIFFERENT METHODS

VINCENZO SAIBENE ***, LUISA BREMBILLA a, ALFRED0 LORENZO BOLOGNANI b and GUIDO POZZA a

BERTOLETTI

a Department of Medical Pathology and b Biological Chemistry, Ospedale San Raffaele, Milan (Italy)

University of Milan,

(Received

September

*,

15th, 1978)

Summary The measurement of glycosylated hemoglobins in diabetic patients represents a new approach to the problem of the long-term glycemic control. Chromatographic procedures are usually employed to separate the glycosylated components of hemoglobin; we performed a comparative analysis of two different methods: chromatographic and calorimetric. Chromatographic separation on small ion-exchange resin columns gives high precision (coefficient of variation = 1.7%) over the whole range of normal and diabetic values of percentage of glycosylated hemoglobin (3.2-15.9%). An alternative technique is the measurement of 5-hydroxymethylfurfural released by oxalic acid hydrolysis of the hexoses bound to hemoglobin, as proposed by Fluckiger and Winterhalter (Fluckiger, R. and Winterhalter, K.H. (1976) FEBS Lett. 71, 356-360). Normal values, expressed as 5-hydroxymethylfurfural absorbance per 10 g of total hemoglobin, range from 133 to 235, with mean ? S.D. = 189 + 26; while diabetic patients show a range from 220 to 443 and mean ?r SD. = 318 + 65. This last method gives satisfactory precision over the entire range of values examined (coefficient of variation = 4.2%) and has proved simple and inexpensive . The correlation between the two methods is very high (n = 20; r = 0.98;~ < 0.001) with a regression line, y = 25.1~ + 40.3. The storage of the hemolysates at -20°C for up to 70 days for the colorimetric method and up to 260 for the chromatographic procedure does not decrease the precision of either technique.

* Correspondence to: Dr. Vincenzo Saibene, Cattedra di Patologia Speckle Medica, Universiti di Milano, Ospedele San Raffaele. 20090 Segrate-Mileno, Itelia.

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Introduction Diabetic patients can show a 2-4-fold increase in the percentage of minor glycosylated components of hemoglobin A, ((Y&) [ 11. The glycosylation is a non-enzymatic reaction occurring slowly during the life-span of the erythrocytes at the NH*-terminal sites of the beta chains: it depends upon the glucose concentrations inside the red cells [ 21. Recent evidence suggests that the percentual concentrations of glycosylated hemoglobin could be related to long-term glycemic control in diabetics: in fact the levels of glycosylated hemoglobins have been reported to be proportional to the entity of glycosuria in the two months preceding their measurement [ 3,4]. Other studies have shown a correlation between the percentage of glycosylated hemoglobins and the peak and mean daily blood glucose levels [ 51, thus supporting the concept that glycosylated hemoglobins represent an integrated index for the glycemic levels. The advantages in using such a t.est instead of sporadic measurements of blood and/or urine glucose concentrations are obvious. Two principal methods have been proposed to measure the levels of glycosylated hemoglobins: isoelectric focusing [6,7] and chromatographic techniques [ 1,8] ; in addition a calorimetric method has been proposed by Fluckiger et al. [9]. In spite of the growing interest in this field, there have been only a few attempts to compare and standardize methods in order to suggest simple and reliable techniques that might be routinely used. In this paper a comparative analysis between two of the proposed techniques for the detection of glycosylated hemoglobins is presented. Methods Hemolysa te preparation 4-8 ml of blood with addition

of EDTA were sampled regardless of the state of feeding or fasting of the subjects. Red cells were separated by centrifugation (1400 X g for 5 min) and then washed three times with saline (9 g/l). Hemolysis was performed by adding to 1 ml of the packed red cells 1.4 ml of distilled water and 0.4 ml of toluene. After rough mixing, toluene and the stromal debris were removed by centrifugation at 1400 X g for 20 min. Further purification was obtained by filtration through Whatmann No. 1 filter paper and by a 12-h dialysis of the hemolysate. If not immediately assayed, the hemolysates were stored at -20°C. Chromatographic

method

Ion-exchange chromatography was performed according to the method of Trivelli et al. [ 11, employing Bio Rex 70 resin of 200-400 mesh (BioRad Laboratories, Richmond, CA, U.S.A.). To shorten the operating time, small columns (7 X 2.5 cm) were obtained from glass 20 ml syringes, as suggested by Kynoch and Lehmann [lo]. 0.5 ml of hemolysate, previously dialyzed against cyanide buffer No. 6 at 4”C, were applied to the top of the resin layer.

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All the glycosylated hemoglobin components (HbA Ia, b, c) were pooled at room temperature in 250 ml of eluted buffer No. 6 and. evaluated by reading absorbance at 415 nm. HbA I(a-c) levels were expressed as percentage of total hemoglobin. 15 samples could be analysed by a single operator in about 3 h. Colorime tric method (Fluckiger and Winterhalter) [l I] Hexoses bound to hemoglobin were quantitatively hydrolyzed by heating at 100°C in presence of oxalic acid. The 5-hydroxymethylfurfural (5-HMF) derivative may be detected by a colour reaction with 2thiobarbituric acid. 2 ml of this hemolysate were added to 1 ml of oxalic acid (0.3 N) in glass tubes and then incubated at 100°C for 60 min. After cooling to room temperature, 1 ml of 40 g/100 ml TCA was added for protein precipitation. This mixture was centrifuged at 1400 X g for 5 min and 2 ml of the clear supernatant were added to 0.5 ml of 2-thiobarbituric acid 0.05 M. After a 40-min incubation at 40°C the absorbance of the samples was read at 443 nm (Bausch and Lomb, Spectronic 100) against a reagent blank. Duplicate samples were assayed for each subject. Total hemoglobin concentration of the hemolysate, previously dialyzed against distilled water, was assessed by the cyanmethemoglobin method (Aculute-Acuglobin; Orthodiagnostic Inc., NJ, U.S.A.) and then adjusted to 10 g/100 ml. Results were expressed as 5-HMF absorbance per 10 g of total hemoglobin. 50 samples could be assayed by a single operator in about 5 h. Usual statistical analyses (mean and standard deviation, linear regression, correlation and variation coefficients, paired and unpaired Student’s t-test) were applied as appropriate. Patients HbA I(a-c) % was assayed by chromatographic method in 33 normal subjects and in 90 diabetic patients. 5-HMF absorbance was determined in 32 normal subjects, 58 diabetic patients and 46 samples of unknown source. 20 samples (4 from normal subjects and 16 from diabetic patients) were assayed by both methods. Normal subjects were healthy volunteers with normal glucose tolerance (100 g OGTT) and without family history of diabetes. Results The mean normal value for HbA I(a-c)% by the chromatographic method was 5.6 t 1.2 (range 3.2-7.8), while diabetic patients showed a mean value of 11.04 f 2.8 (range 3.7-15.9). The difference between the two groups was highly significant (p < 0.001). The mean normal value for 5-HMF absorbance per 10 g of total hemoglobin was 189 + 26 (range 133-235); the mean value of the examined diabetic was

202

TABLE

I

COEFFICIENTS ODS

FOR

STORAGE

OF

VARIATION

(C.V.)

GLYCOSYLATED AT

OF

COLORIMETRIC

HEMOGLOBIN

AND

DETECTION

CHROMATOGRAPHIC

ACCORDING

TO

THE

METHLENGTH

OF

-20%

Number

Time

of

at -20°c

samples

of storage

C.V. Partial

(days)

Total

Calorimetric 33

0

4.34

31

S-15

3.26

29

16-30

4.92

27

31-60

4.28

16

>60

4.2

3.67

Chromatographic 9 10

0 150-260

1.64 1.74

1.7

318 k 65 (range 220-443) with a highly significant difference vs. normal (p < 0.001). The coefficient of variation (C.V.) for the chromatographic method was 1.7%; C.V. for the calorimetric method was 4.2% (Table I). The storage at -20°C of the hemolysates for up to 260 days for the chromatographic method and up to 70 days for the calorimetric determination did not decrease the precision of either procedure, as shown in Table I. In fact there was no significant difference among the partial C.V. for different lengths of time of storage, neither was there any correlation between intra-assay standard deviation and the duration of the storage (r = -0.06 for the chromatographic method and r = 0.1 for the calorimetric method). Moreover, no significant difference was evident by chromatographic assay of 10 samples before and after storage for up to 260 days (t = 0.58;~ = n.s.).

123456'78

9

HbAI (a-c)% Fig. HbA

1.

Correlation

I(a--c)J

between

(chromatographic

(chromotographic

5-HMF

absorbance

method)

10

11

12

13

14

15

16

method) (A)/10

in 20 subjects.

g of

total

hemoglobin

(calorimetric

method)

and

203

Neither method showed any significant change in precision over the range of values examined: there was no correlation between intra-assay standard deviation and intra-assay mean values (r = -0.05 for chroma~~aphi~ method and P = 0.17 for calorimetric method). Testing the samples with both methods evidenced a strong correlation between HbA I(a-c)% and 5HMF absorbance per 10 g of total hemoglobin (Fig. 1) (r = 0.98;p < 0.001). The regression formula is: y = 25.1~ + 40.3, where y = 5-HMF absorbance per 10 g of total hemoglobin and IX:= HbA I(a--c)% Calculation by this formula of HbA I(a-c)% from the 5-HMF absorbance per 10 g of total hemoglobin data showed normal mean values and a range fully comparable with HbA I(a-c)% obtained by the chromatographic procedure (5.9 + 1; range 3.7-78 vs. 5.6 t 1.2; range 3.2-7.8). Discussion Recent observations suggest the importance of glycosylated hemoglobin detection for the evaluation of long-term glycemic control in diabetic patients [12-141. However, the practical use of this parameter has been limited until now by the complexity of the techniques required. Several methods have been proposed for the preparation of hemolysates [ 1,10,14-161; the usual technique, described in this paper, is time-consuming, yet a high purification of the hemolysate may be necessary. In fact the absence of stromal debris allows repeated use of the columns with obvious economic advantages; the calorimetric method requires several washings of the red cells and dialysis of the hemolysates to remove glycosylated peptides of non-hemoglobin origin (glycoproteins, glycosylated stromal debris) which may constitute an unspecific source of 5-HMF. Prolonged storage of the hemolysates at -20°C does not interfere with the precision of either method and this may be of practical relevance: however, for the chroma~~aphic technique prior dialysis against cyanide buffer is required to stabilize the prosthetic heme group. Electrophoretic methods [17] have also been employed for assay of glycosylated hemoglobins; however, their separation from HbA is not satisfactory even by isoelectric focusing. In fact the isoelectric points of HbA and HbA Ic differ by only 5 hundredths of a pH unit. Recently, Beccaria et al. (1978) 17 ] have employed a pH-~adient modifier, the dipeptide His-Gly which allows a better separation between the two fractions. However, the technical difficulties involved in this method do not permit its routine clinical application. Chromatographic techniques present no difficulties for separating the glycosylated components: these are eluted before the main peak of HbA and for this reason they have been called “fast hemoglobins”. Drawbacks of these methods are the length of time required and the use of cyanide buffers. Both these difficulties are limited by using small columns and by pooling the minor fractions. HbA Ia and HbA Ib contain, in their molecule, hexoses (glucose-6-P, fructose1,6-P and still unidentified carbohydrates) [ 18,191 and their levels are increased in diabetic patients [3,13,15,19] : their collection together with HbA Ic seems therefore correct.

204

Recently, attempts to shorten further the time required for chromatographic analysis have been made: among them the use of microcolumns [14,15] and high pressure liquid chromato~aphy [l&16]. The mean normal value obtained by the microchromatographic technique, by Welch and Boucher [ 141, is rather higher than in previously reported data, with attendant overlapping between normal and diabetic ranges; more sophisticated separating procedures such as high pressure liquid chromatography, despite the reduced elution time (a few minutes), are less precise (C.V. from 5 to 10%) [ 161. Furthermore, the complexity of the apparatus required prohibits routine use of this method. A quite new approach comes from the work of Fluckiger and Winterhalter [ll] : these authors show that hydrolysis by oxalic acid of HbA Ic releases 5-HMF, which can be determined calorimetrically. Our data show a significant difference between normal and diabetic mean values of 5.HMF absorbance, with minor overlapping between the two groups. The very high correlation between HbA I(a--c)% and 5-HMF absorbance suggests that ‘a’ and ‘b’ fractions form a negligible share of the eluted HbA I(a*). In this connection, while previous reports considered HbA Ia and HbA Ib levels to be 1.6 and 0.8% [8], more recent data suggest 0.8 as a suitable figure for their percentage of total hemoglobin [ 201. Such a high correlation may also be explained by the presence in HbA Ia and HbA Ib fractions of hexoses, detectable by the calorimetric reaction of Fluckiger and Winterhalter [II], and concomitantly increased in diabetic patients [ 181. According to our regression formula between 5-HMF absorbance and HbA I (a-c) percentage it is conceivable that acid hydrolysis may release 5-HMF from hexoses bound to sites different from HbA I(a---c) fractions; in fact the y intercept for HbA I(a-c)% = 0 is 40. This observation agrees with prelimina~ reports of Bunn et al. [ 181, who show the presence of sugars by Fluckiger and Winterhalter’s reaction in the chromatographic peak of the major hemoglobin component. The calorimetric method exhibits a higher C.V. than the chromatographic technique (4.15% vs. 1.69%): however, some advantages are evident. The method is inexpensive and simple: no special equipment is required, more samples may be assayed at the same time and no cyanide has to be employed. In some circumstances, such as the presence of HbF, where fetal hemoglobin cannot be separated by chromatography from HbA Ic, the calorimetric method must be used. Results of calorimetric method may be expressed as “5-HMF absorbance per 10 g of total hemoglobin” concentration. Alternatively our regression formula may be applied to convert 5-HMF absorbance to HbA I(a-c)%. The advantage of the chromatographic method described is its high precision (C.V. = 1.7): when extraordinary accuracy is required, it could be the method of choice. Acknowledgements Skilful edged.

technical

assistance

of Mr. Angelo

Carenini

is gratefully

acknowl-

205

References 1 Trivelli, 2 Koenig,

L.A., Ranney,H.M.and Lay.H.-T. (1971) N. Engl. J. Med. 824.353-357 R.J. and Cerami, A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72. 3687-3691

3 Gabbay, K.H., Hasty, K.. Breslow. J.L., Ellison, R.C.. Bunn, H.F. and Gallop. P.M. (1977) J. Clin. Endocrinol. Metab. 44.859-864 4 Lanoe, R.. Soria, J., Tbibult, N.. Soria. C.. Escbwege, E. and Tcbobroutsky, G. (1977) Lancet ii, 1156-1157 5 Gonen, B., Rubenstein, A.H.. Rochman. H., Tanega, S.P. and Horwitz. D.L. (1977) Lancet ii, 734737 6 Krishnamoorthy, R., Wajcman. H. and Labie, D. (1976) Clin. Chim. Acta 69. 203-209 7 Beccaria, L., Chiumello. G., Gianazza, E., Luppis. B. and Righetti. P.G. (1978) Am. J. Haematol. 8 9 10 11 12 13 14 15 16 17 18 19 20

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