A COLORIMETRIC METHOD FOR THE ESTIMATION OF SERUM GLYCATED PROTEINS BASED ON DIFFERENTIAL REDUCTION OF FREE AND BOUND GLUCOSE BY SODIUM BOROHYDRIDE

CHAPTER V A COLORIMETRIC METHOD FOR THE ESTIMATION OF SERUM GLYCATED PROTEINS BASED ON DIFFERENTIAL REDUCTION OF FREE AND BOUND GLUCOSE BY SODIUM BOROHYDRIDE

132 INTRODUCTION The measurement of glycated hemoglobin has been widely used as an index in assessing long-term diabetic control, which reflects the glycemic status prevailing over the preceding two to three months(chapter I, Pages 45-50). However, it is not considered to be a sensitive indicator of shortterm (2-3 weeks) glycemic control to monitor the rapid changes in glycemic status that occur with alteration in treatment (393). Glycated albumin and glycated serum proteins are suggested to be better parameters for this purpose (Chapter II, Pages 51-53). Glucose bound nonenzymatically to serum proteins can be assayed as 'fructosamine* which is based on the reducing activity of bound sugars (ketamines) tinder cold alkaline conditions (251) or by estimating hydroxymethyl furfural released on mild acid hydrolysis of the proteins, by complexation with thiobarbituric acid(9,241). The data presented in Chapter IV (Figure 10, Page 121) indicate that sodium borohydride at low concentrations can reduce fructose and glucose completely as evidenced by the abolition of chromogen formation in the phenol-sulphuric acid reaction, whereas glycated hemoglobin and glycated albumin require much higher concentration of borohydride for reduction of comparable amounts of glyco groups. It has been shown that purified albumin was fully reduced by 20 mg sodium borohydride(390), whereas an equivalent amount of free glucose was reduced by less than one mg

133 of the reductant(chapter IV, Figure 10, Page 121). This differential action of sodium borohydride on free glucose and on glucose bound nonenzymatically to proteins is made use of in developing a simple colorimetric method for the estimation of serum glycated protein, based on phenolsulphuric acid reaction. The details of the procedure and the relative merits of glycated protein values and glycated albumin levels in evaluating hyperglycemia are presented in this chapter. MATERIA 15 Blood samples from healthy subjects(20-40 yr), diabetics (20-60 yr) and subjects suspected of diabetes (40-60 yr) were collected, and processed as described in Chapter IV(Page 105). Other materials used are listed in Chapter II (Page 67). METHODS Determination of NaBH^ reducibility of free and bound sugars: Extent of NaBH^ reduction was studied as follows. To 0.02 ml of serum or sugar solution, 0.2 ml of freshly prepared aqueous solution (containing two drops of ammonium hydroxide per ml) of NaBH4 (final concentration varying from 1-30 mg) was added. After 15 min standing at room temperature(28#- 30*C), the volume was made upto 1.0 ml and phenol-sulphuric acid color reaction was performed as described in Chapter II

134 (page 72). Apart from a reagent blank (without phenol solution), blanks with appropriate concentrations of NaHH4 (added after addition of sulphuric acid) were run. This was found to be necessary, since NaBH4 'per se' depressed color intensity of carbohydrates in phenol-sulphuric acid reaction. For example, NaBH^ addition depressed color intensity of glucose by 11.3% at 2 mg level and by 20.0% at 20 mg level. Determination of serum glycated proteins bv differential borohydride reduction: Hexose bound to serum proteins nonenzymatically was estimated as follows. To 0.02 ml aliquots of serum samples, 2 and 20 mg of NaBH4(in 0.2 ml) were added. After 15 min standing the volume was made to 1.0 ml with water followed by phenol solution(0.05 ml 80% phenol) and concentrated sulphuric acid (3.0 ml). The difference in color intensities measured after 30 min at 480 nm between the two systems was a measure of glycated proteins. The values were expressed as glucose equivalents. In some studies, serum proteins were prepared as follows. To 0.2 ml serum, 0.2 ml of 10% trichloroacetic acid was added. The precipitate was washed with 5% trichloroacetic acid until the washings did not show positive reaction in phenol-sulphuric acid reaction. The precipitate was dissolved in 0.2 ml and reducible bound hexose was estimated as described above using 20 mg NaBH4.

135 Determination of glycated albumins Albumin from serum was isolated by chromatography on Blue Sepharose and total and reducible bound hexose were determined by phenol-sulphuric acid method described in Chapter II (Page 76). Determination of glycated hemoglobin: Globin bound hexose was estimated by the method detailed in Chapter II(Page 67). Blood glucose was estimated by O-toluidine method(375). Oral glucose tolerance test was performed with a load of 50 g glucose. Total serum protein was estimated by biuret method(394) and protein in chromatographic fractions was determined by the method of Lowry et al(374). RESULTS To check the differential efficacy of Naffl^, studies on reduction of sugars and serum were undertaken. In Figure 13 (Page 136) the magnitudes of reduction(measured as a decrease in color intensity of phenol-sulphuric acid reaction) of monosaccharides, and disaccharides as a function NaBH^ concentration are represented. The data indicate that glucose(100 pg in 20 pi) was completely reduced

136 PERCENT REDUCTION FIGURE 13 - REDUCTIBILITY OF FREE SUGARS AS A FUNCTION OF SODIUM BOROHYDRIDE CONCENTRATION. A - Glucose* 100 pg(e ), 200 figo... ), 400 fig(0--------o) and 100 fig at 4 C{0-------- 0), Maltose, 100 fig(x--------x). B - Galactose, 100 fig(* ), 100 fig at 4 C(B -- ), Lactose (X--------X).

137 by 0.5-1.0 mg NaBH4 at 28-30 C in 15 min as evidenced by the disappearance of color in phenol-sulphuric acid reaction. For the complete reduction of 200 pg glucose(55 mm) and 400 pg glucose(110 mm), respectively 2-3 mg and 3-4 mg of NaBH4 were adequate. Galactose was found to behave similarly. The reduction of both glucose and galactose was less efficient at 4 C compared to room temperature. Lactose and maltose at 100 pg levels were reduced to the extent of 48-50% by 2 mg NaBH^. Increasing the concentration of the reductant to 20 mg, caused only a marginal increase(2.0%) in reduction as indicated by decrease in phenol-sulphuric acid reaction color intensity. The data suggest that the reducing ends in monosaccharides and disaccharides are efficiently converted to the alcohols by low concentrations of NaBH4 and the glycosidic linkages in oligosaccharides are virtually unaffected even by high concentrations of the reducing agent under the experimental conditions. In Figure 14(Page 138), the effect of NaBH4 reduction on phenol-sulphuric acid positive reactants in serum(undialyzed and dialyzed) as such and after fortification with glucose is represented. With dialyzed serum, NaBH4 (2 mg) reduced the color intensity only to a small extent(2.0%), whereas with native serum there was a sharp fall in color intensity(35%) accounting for 61.0 mg/dl of glucose. Similarly, in the case of serum to which glucose was added externally to the extent of 100-200 jig, the color intensity in phenol-sulphuric acid

138 ABSORBANCE VALUES (X 460) FIGURE 14 - REDUCIBILITY OF PHENOL-SULPHURIC ACID POSITIVE REACTANTS BY NaBH^ IN 0.02 ml OF NATIVE SERUM (A), DIALYZED SERUM(X), SERUM FORTIFIED WITH 100 fig OF GLUCOSE ( ) AND SERUM FORTIFIED WITH 200 fig OF GLUCOSE(O).

139 reaction after reduction with 2 mg of NaBH^ decreased to levels comparable with dialyzed serum values. These results show that blood glucose levels upto concentrations as high as 55 mm will be effectively reduced by 2 mg of NaBHj. In the range of 2-4 mg, NaBH^ did not cause any further decrease in color intensity. Further increase in NaBH^ concentration caused a decrease in values upto a concentration of 20 mg of the reductant. The absorbance values observed with 30 mg of NaBH4 was not significantly lower compared to absorbance observed with 20 mg of the reducing agent. The data suggest that the difference in the amount of phenol-sulphuric acid positive material assayed with 2.0 mg of the reductant and without the reductant will be a measure of free glucose, whereas the difference observed with 2.0 mg and 20.0 mg of NaBH4 will represent the glycated proteins and glycated peptides in serum. To ascertain whether transient fluctuations of blood glucose will affect the determination of hexose bound nonenzymatically to serum proteins, the differences in color intensity in phenol-sulphuric acid reaction after reduction with 2 mg and 20 mg of NaBH^ were estimated in 5 diabetic patients during oral glucose tolerance test. The results are shown in Table 18 (Page 140). Even though blood glucose levels increased by 2.1-2.6 fold at 1-2 hr compared to values at 0 hr, bound hexose values changed only to the extent of -3.4% to -1-3.5% during the test. The changes were well within

140 TABLE 18 LEVELS OP GLYCATED PROTEIN IN PRE- AND POST-PRANDIAL SERUM OP DIABETIC PATIENTS No Time (hr) Blood glucose (mm) Serum glycated protein (pg hexose/mg protein) 0 6.2 9.60 1 1 2 11.6 13.0 9.94 9.94 0 2 1 2 10.9 19.2 22.5 10.2 10.2 10.2 0 3 1 2 9.5 22.0 20.3 9.80 9.80 9.80 0 4 1 2 8.3 14.8 21.4 9.5 9.5 9.3 0 11.7 10.2 5 1 18.3 9.9 2 23.2 10.2

141 the coefficient of variation for the method( + 3.8%, n = 6). That the amount of glycated proteins estimated by the differential reduction with NaBH4 is a true reflection of glucose bound nonenzymatically to serum proteins, is supported by the comparison of bound hex os e estimated in serum as such and in proteins precipitated by trichloroacetic acid in sexrum. The values in individual serum samples are correlated in Figure 15(Page 142). The correlation coefficient was very high(r» 0.99, PC0.001). The consistently lower values (to the extent of 6.3-10.7%) in the precipitated proteins compared to native serum could be due to the solubility of glycated peptides and some glycated proteins in trichloroacetic acid. The protein bound hexose values(differential reduction with 2 mg and 20 mg of NaEH^) in normal and diabetic serum samples are represented in Figure 16 (Page 143) and Table 19 (Page 144). The values are given both in terms of pg hexose/mg protein and mg hexose/ml serum. The values in terms of pg hexose/mg protein in control subjects (n = 32) and and diabetics (n * 50) were respectively 5.06 + 0.37(Mean + SD) and 11.0 + 1.77 and the increase in the latter group was statistically significant(p< 0.001). Similarly, the increase in the values of mg hexose/ml serum in diabetics(0.760 + 0.13) was significant compared to the values in control subjects (0.415 + 0.05). However, the mean increase of the former parameter was 2.17 fold and slightly higher than the increase

142 BOUND HEXOSE, )ig IN PRECIPITATED PROTEIN BOUND HEXOSE, [JG/0 02 ML SERUM FIGURE 15 - CORRELATION BETWEEN BOUND HEXOSE VALUES IN SERUM(O.D. DIFFERENCE WITH 2.0 mg AND 20.0 mg NaBH4) AND IN PRECIPITATED SERUM PROTEINS(O.D. DIFFERENCE WITH 0.0 AND 20.0 mg NaBH^). NORMALS ( ) AND DIABE TICS (0).

143 MICROGRAM HEXOSE/ MILLIGRAM HEXOSE/ MG PROTEIN ML SERUM IZJ 12 - : * * t : : t* i i: :!. #* «. 10 0*8 0-6 0-4 0-2 CONTROL DIABETIC CONTROL DIABETIC 0 FIGURE 16 - GLYCATED PROTEINS IN SERUM BY DIFFERENTIAL NaBH. REDUCTION. 4

144 TABLE 19 LEVELS OF SERUM GLYCATED PROTEIN IN NORMALS AND DIABETICS Normals(n = 32) Mode of expression Mean + SD (range) Diabetics(n = 50) Mean + SD (range) jig Hexose/mg 5.06 + 0.37 11.0 + 1.77 protein (4.35-6.08 (6.45-14.84) pnol Hexose/mg 0.028 + 0.002 0.061 + mm 0.009 protein (0.024-0.033) (0.035-0.082) mg Hexose/ml 0.415 + 0.05 0.760 + 0.13 serum (0.333-0.540) (0.500-1.02) mmol Hexose/1 2.30 + 0.27 4.22 + mm 0.72 serum (1.85-3.00) (2.77 5.66)

145 in the latter(1.83 fold). The discrimination between diabetic and control groups was better if hexose bound nonenzyraatically to serum proteins was expressed as a function of protein concentration. This is because of the lowered concentration of serum total proteins in diabetics (6.95 + 0.95 g/dl Mean + SD) compared to the values in control group (8.02 + 0.40 g/dl). Thus, for routine purposes, expression of glycated proteins levels per unit volume of serum may be adequate to detect hyperglycemia, since this avoids the additional assay for serum proteins. Total hexose bound(nonenzyraatic and enzymatic) to serum proteins can be calculated from the color intensities in phenol-sulphuric acid reaction produced by serum after prior treatment with 2 mg of NaEH^. The values in control subjects (n * 20) and diabetic patients (n =* 25) were found to be 1.10 + 0.01 (Mean + SD) and 1.51 + 0.18 mg/nl serum respectively. Even though the difference was significant (PCQ.OOl) the mean increase(1.37 fold) in diabetic group was much lower than the increase found in the case of hexose bound nonenzymatically. Prom the values for the total bound hexose and hexose bound nonenzymatically, the levels of carbohydrates in glycoproteins in the serum can be assessed. The values in control subjects(n a 20) and diabetics (n = 25) were respectively 0.64 + 0.12 (Mean + SD) and 0.64 + 0.15 mg hexose/ml serum. The correlation between glycated serum protein levels measured by the differential borohydride reduction and fasting

146 blood glucose values was highly significant(r * 0*77, P< 0.001) as indicated in Figure 17 (Page 147). However# the relationship between post-prandial (2 hr) blood glucose and glycated proteins was not significant(r» 0.35#P<0.05# n a 25# data not shown). In Figure 18(Page 148) glycated serum protein values are correlated to total glycated serum albumin levels and NaBH4 reducible glycated albumin levels. Good correlation between glycated serum proteins and albumin values(r * 0.85 for total# r * 0.88 for reducible glycated albumin) was observed suggesting that the estimation of hexose bound nonenzymatically to serum proteins will be as reliable as estimation of glycated albumin in the detection of hyperglycemia Serum glycated protein levels and glycated albumin levels (total and reducible) were determined in patients undergoing glucose tolerance test and who were suspected to have impaired glucose tolerance or preclinical diabetes. The results are shown in Table 20(Page 149). The patients were categorized into three groups based on the relative degree of impairment of glucose tolerance. Group I(n * 5) consisted of subjects with normal fasting blood glucose level and relatively mild impairment. Group II(n m 7) consisted of patients with normal or slightly elevated fasting blood glucose but elevated 2 hr post-prandial glucose level but all within renal threshold. Group III(n * 8) comprised

147 FASTING BLOOD GLUCOSE VALUES, m M. PROTEIN BOUND HEXOSE,pG/MG PROTEIN FIGURE 17 - CORRELATION BETWEEN GLYCATED SERUM PROTEIN LEVELS AND FASTING BLOOD SUGAR VALUES.

148 GLYCATED ALBUMIN MOL HEXOSE / MOL PROTEIN BOUND HEXOSE [ig / MG PROTEIN FIGURE 18 - CORRELATION BETWEEN GLYCATED SERUM PROTEIN LEVELS AND GLYCATED ALBUMIN LEVELS. TOTAL GLYCATED ALBUMIN( ). NaEH4 REDUCIBLE GLYCATED ALBUMIN(O).

LEVELS OF GLYCATED SERUM PROTEINS, GLYCATED ALBUMIN AND BLOOD GLUCOSE IN SUSPECTED DIABETICS Parameters Control Mean+SD Confirmed diabetics Mean + SD Group I Mean+SD Suspected Diabetics Group II Mean+SD Group III Mean + SD Fasting blood glucose mm 4.7 + 0.3 5.0 + 0.5 6.1 + 0.06 Post-prandial blood glucose mm (2 hr) 5.8 + 1.2 8.8 + 0.7 13.0 + 2.5 Serum glycated protein pg hexose/mg protein 5.06 + 0.37 11.0 + 1.77 8.78 + 0.37 9.49+ 1.22 9.60+ 1.06 Total glycated albumin mol hexose/mol protein 1.66 + 0.23 3.59 + 0.67 2.74+ 0.13 2.79+ 0.39 2.87+ 0.28 Reducible glycated albu- 1.24+0.02 2.53+0.43 min mol hexose/ ~ mol protein 2.05+ 0.11 2.16+ 0.38 2.16+ 0.31

150 patients with elevated fasting blood glucose and post-prandial blood glucose(2 hr) above renal threshold. The serum glycated protein values were found to be increased significantly (PC0.001) in all 3 groups of subjects compared to controls. The mean increase was 1.73, 1.87 and 1.90 fold in groups I, II and III respectively. However, there was no significant * difference among the individual groups. Similar pattern could be observed with reference to total glycated albumin and reducible glycated albumin. The mean increase compared to controls in the individual groups were 1.65 and 1.65(group I) 1.68 and 1.74(group II) and 1.72 and 1.74(group III) respectively for total and reducible glycated albumin. The correlation between glycated serum proteins and albumin values were highly significant^ * 0.81 for total, r» 0.89 for reducible glycated protein. Figure 19, Page 151). The glycated hemoglobin levels in 10 randomly selected cases were found to be in the range 0.349-0.510 mol hexose/nol protein(0.423 + 0.044, Mean + SD) The mean increase was 1.37 fold in subjects with elevated 2 hr post-prandial blood glucose (below renal threshold level) and 1.58 fold in subjects with 2 hr post-prandial blood glucose (above renal threshold level) compared to normals. DISCUSSION In this chapter a method for estimation of serum glycated proteins based on differential reduction of free glucose and

151 GLYCATED ALBUMIN, MOL HEXOSE/MOL PROTEIN BOUND HEXOSE JG/MG PROTEIN FIGURE 19 - CORRELATION BETWEEN GLYCATED SERUM PROTEIN LEVELS AND GLYCATED ALBUMIN LEVEL IN SUSPECTED DIABETICS. Total glycated albumin : Group 1(A), Group 11(D) and Group III(O). Reducible glycated albumin : Group 1(A), Group II( ) and Group!!!( ).

152 glucose bound nonenzymatically to protein with 2 and 20 mg NaBH^ employing phenol-sulphuric acid color reaction is described. It is evident from the present study that free glucose in the serum upto concentrations as high as 55 mm can be completely reduced by 2 mg of NaHH^, whereas glucose bound nonenzymatically is virtually unaffected. Use of 20 mg of NaBH^ was found to reduce a finite amount of hexose which represents the glycated proteins since NaEH4 was found to have no effect on glycosidic links under similar conditions. Early workers reported an increase in serum protein bound hexose values in a variety of pathological conditions including diabetes (395). When total protein bound hexose levels were measured in the serum after selective reduction of free glucose by NaHH^, using the phenol-sulphuric acid color reaction, a mean increase of 1.37 fold in the values in diabetics was observed in the present study. The values in control subjects were found to be comparable to the protein bound hexose values determined by orcinol-sulphuric acid method(395). However# the levels of glycosidically bound hexose values(after reduction of ketamine linked hexose by 20 mg of NaBH^) did not increase in the diabetic group indicating that the increase of bound hexose observed is exclusively due to glycated proteins. A mean increase of 1.83 fold of glycated protein in serum was observed in diabetic patients by the new colorimetric procedure described here when the values were expressed in terms of serum volume. This compares favourably with the reported elevation of 1.3 fold and 1.39 fold in serum

153 fructosamine values in diabetes(396,397). Serum glycated protein estimated by the thiobarbituric acid assay is reported to be elevated 2.25 and 2.18 fold(239,241) when the values were expressed as a function of protein concentration which agrees well with the increase in serum glycated proteins in diabetes(2.17 fold in terms of protein concentration) reported by the present method. Recently Go and associates (259) showed a 3 fold increase in serum glycated protein in diabetics by an enzyme-linked immunosorbent assay. The increase in serum glycated proteins observed in the present study also correlated well with the elevation of glycated albumin (2.09 fold total, 2.26 reducible) estimated by phenol-sulphuric acid reaction in diabetics(382). In addition, it was observed that glycated proteins determined by the differential borohydride reduction correlated well with fasting blood glucose values and glycated albumin levels in individual diabetic cases. McFarland and associates(9) reported glycated protein values of 0.63-1.31 nmoles of HMF/mg protein in normals and 1.54-3.97 in diabetics by thiobarbituric acid method. Kennedy et al(239) who employed a prior dialysis step to avoid the interference of serum glucose in thiobarbituric acid method reported values of 0.32 and 0.72 nmoles HMF/mg protein for normals and diabetics respectively. Murtiashaw et al(241) who employed a prior ethanol precipitation of serum to avoid the interference by endogenous glucose in thiobarbituric acid method reported essentially similar values(0.38 +.0*10 nmol

154 HMF/mg protein for normals and 0.83 + 0.18 for the diabetics) of serum glycated protein. Elder and Kennedy(398)have reported a value of 1.5 + 0.058 nmol fructose/mg protein for normals and 3.3 + 0.145 for diabetics employing a modified thiobarbituric acid method. The present values of serum glycated protein (20 + 2 nmol hexose/mg protein in normals and 61 + 9 in diabetics) are much higher than the reported levels of serum glycated protein by thiobarbituric acid method. High values for glycated albumin(382) and glycated hemoglobin(244) have been reported using the phenol-sulphuric acid method compared to thiobarbituric acid method. It has been reported that the amount of hydroxymethyl furfural formed by thiobarbituric acid method accounts for only a small proportion of total bound hexose nonenzymatically( 79 ). This may partly explain the low values in thiobarbituric acid method. Baker et al(396) and Lloyd and Harpies(397) reported serum fructosamine levels as 2.26-5.22 m moles/1 and 2.17 + 0.09 m moles/1 respectively in diabetics. The present values of 4.22 +0.72 mmoles/1 of glycated protein in diabetics determined by an entirely different principle are comparable with the reported levels of Baker et al for fructosamine(396) However, serum glycated proteins reported by any of these methods cannot be considered as absolute values. In the fructosamine method deoxymorpholinofructose is commonly used as standard based on the assumption that reducing activity of this standard and glycated proteins are comparable. Johnson and Baker have

155 underlined this aspect in a recent communication(399). In the present method glucose is used as standard. There is no reason to expect that the nature of the chromogens and their quantities formed from glycated protein and glucose are same. Schleicher et a1(79) have shown that glycated proteins on treatment with concentrated mineral acid produce furoylmethyllysine apart from hydroxymethyl furfural. Chang and associates (119) identified 2-furoyl 2-furanyl imidazole as a product of glycation of proteins. The relative intensities of chromogen formed by these compounds in phenol-sulphuric acid reaction by glycated protein are not known. All the cases of impaired glucose tolerance cannot be discerned on the basis of glycated hemoglobin measurement (Chapter I, Pages 40-45) In this regard, serum glycated proteins have been suggested to be sensitive enough to distinguish impaired glucose tolerance(9 ). The sensitivity of glycated serum protein in detection of impaired glucose tolerance was evaluated. The data showed that both glycated serum protein and serum glycated albumin are useful in identifying most cases of impaired glucose tolerance. However, subjects with mild impairment of glucose tolerance and those with diabetic like tolerance could not be discerned. It may be concluded that serum glycated proteins may serve as better indicators of impaired glucose tolerance compared to glycated hemoglobin (which is increased only marginally in subjects with mild impairment of glucose tolerance and values overlap with control values).

156 However# the present method can conveneiently be used for the determination of total serum glycated proteins in detection of diabetes. Its efficacy in monitoring glyceraic control during treatment is yet to be evaluated. The method does not require rigorous maintenance of ph and temperature required in the fructosamine assay. It is simple compared to the thiobarbituric acid method which requires precipitation of serum proteins and selective extraction of the chromogen in organic solvent to get reliable values. o