An interspecies comparison of normal levels of glycosylated hemoglobin and glycosylated albumin

Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 819-822, 1985 Printed in Great Britain

0305-0491/85 $3.00+ 0.00 © 1985PergamonPress Ltd

A N INTERSPECIES COMPARISON OF N O R M A L LEVELS OF GLYCOSYLATED HEMOGLOBIN A N D GLYCOSYLATED A L B U M I N MARC RENDELL*, P. M. STEPHENt, ROBERT PAULSENt, J. L. VALENTINE~,KATHY RASBOLD*, TIM HESTORFE*, SUSAN EASTBERG~and DANIEL C. SHINTt *Diabetes Institute and tDepartment of Pharmacology, City of Faith Medical and Research Center, The Oral Roberts University School of Medicine, Tulsa, OK 74170, USA (Tel: 918-493-1000) (Received 5 December 1984)

Abstract--1. Aminophenylboronic acid affinity chromatography was used to measure glycosylated hemoglobin and glycosylated albumin levels in a variety of species. 2. The highest glycosylated hemoglobin levels were found in man, the lowest in the chicken and the pig. 3. The highest glycosylated albumin levels were found in avian species, the lowest in the mouse and the rat. 4. A simple kinetic model was used to analyze the rates of formation of glycosylated hemoglobin and albumin in the various species. 5. Rates of glycosylated albumin formation were very similar across the species while rates of glycosylated hemoglobin formation were quite different, presumably reflecting wide differences in erythrocyte permeability to glucose among the species.

effects of red cell permeability to glucose and changes in red cell physiology may reflect on glycohemoglobin levels (Smith et al., 1982). Such effects would not be expected to modify glycoalbumin formation. Glycohemoglobin levels in a number of species have been studied in the past (Yue et al., 1982) and have been found to be most profoundly influenced by the glucose permeability of the erythrocyte (Higgins et al., 1982). Glycohemoglobin levels are highest in the primates, corresponding to a high degree of glucose permeability. In the rabbit and .the dog, glycohemoglobin levels are lower, in accord with less erythrocyte permeability, and glycohemoglobin levels are lowest in the pig, where glucose permeability is also extremely low. There has been no previous attempt to quantitate glycoalbumin levels in various animal species. We proceeded to compare glycohemoglobin and glycoalbumin levels in a variety of animal species in order to assess the effects of varying erythrocyte physiology and glucose permeability on glycohemoglobin levels in contrast to glycoalbumin levels.

INTRODUCTION

Aminophenylboronic acid affinity chromatography has proved to be an excellent technique for measurement of glycosylated hemoglobin. Results are comparable to those obtained by colorimetric, dectrophoretic, and ion exchange methods (Mallia et al., 1981; Yue et al., 1982a). However, the affinity technique is much less affected by "labile" glycohemoglobin and other interfering factors than the other techniques (Klenk et al., 1982). Furthermore, variations in pH and temperature have a relatively minor effect on affinity chromatography as opposed to ion-exchange chromatography (Klenk et al., 1982). The procedure is also far less labor intensive than colorimetric and electrophoretic methods. Recently we have developed a simple affinity procedure for the quantitation of glycosylated plasma albumin (Rendell et al., 1985a). This technique measures true ketoamine linked glucose and is unaffected by many non-specific factors which interfere with thiobarbituric acid determinations (Rendell et al., 1985b). The ability to reliably measure glycosylated albumin could potentially be an important supplement to glycosylated hemoglobin measurement in the management of diabetes. The half life of human serum albumin is much shorter (about 14 days) than that of hemoglobin (red cell life span 120 days). Therefore, glycosylated albumin levels can potentially furnish an intermediate term perspective on blood sugar control whereas glycohemoglobin levels reflect blood sugar levels over a 2 to 3 month prior period. Albumin is freely accessible to glucose in the blood whereas hemoglobin is enclosed in erythrocytes. The

METHODS

Blood samples Specimens from non-diabetic animals were obtained by venipuncture or cardiac puncture and collected into EDTA evacuated blood collection tubes. Hemolysates were prepared from unwashed packed cells by lysing 1 vol of cells with 20 vols of distilled water. After centrifugation (800g, 10 min) to settle cell debris, the supernates were frozen and stored at -20°C for as long as a week or -70°C for as long as 6 months pending analysis for glycohemoglobin. Plasma samples were centrifuged again (800g, 10rain) prior to similar removal of supernatant, freezing and storage.

819

820

MARC RENDELLet al.

Measurement o f glycoslated hemoglob&

The affinity chromatographic technique was used as previously described (Klenk et al., 1982) using ½ml columns of m-aminophenylboronic acid agarose (Glycogel, Pierce Chemical Company, Rockford, IL).

simple mathematical model. The concentration of glycoalbumin in the blood is describable by a differential equation allowing for the formation of glycoalbumin (GA) from glucose (G) and albumin (A) and a steady rate of degradation of glycoalbumin:

d[GA]

Affinity measurement o f glycosylated albumin As previously described (Rendell et al., 1985a,b), 1 ml

plasma samples were premixed with 4 ml of wash buffer (250mmol/1 ammonium acetate, 50mmol/l MgC12, 0.2 g/1 sodium azide, pH 8.0) in order to equilibrate pH and ionic strength for addition to the column. The resulting 5ml solution was applied to 1 ml m-aminophenylboronic acid agarose (Glycogel, Pierce Chemicals) columns previously equilibrated with wash buffer. After an additional 15ml wash, bound albumin was eluted with 3 ml of elution buffer (sodium citrate, pH 4.5). Albumin in the wash (non-bound) and eluted (bound) fractions were then determined by reaction with bromcresol green (A630) after subtraction of appropriate column blank values or by UV absorbance (A280). Determination of percent bound albumin was then made by the formula: 300 × A bound fract. Per cent = 20 x A non-boundfract. + 3 x A bound fract.

dt

- kl[G] [A] - k2[GA].

The percentage of glycoalbumin equals glycoalbumin concentration divided by total albumin concentration: d([GA]/[Tot Alb]) dt

= kl[G]

([Tot Alb] -- [GA]) [Tot Alb]

[GA] k2 [TotAlb]' [GAI [Tot Alb]

k~

[G] e (kl[G]+k2)t (k,[G] + k2) [G] + k~ (kt[G] + k2)'

So,

at

steady-state,

glycoalbumin

[GA]

The animals used in this study included Sprague-Dawley rats, BALB/c mice, New Zealand white rabbits, gerbils, dogs, pigs, chickens, turkeys and ducks. Values for glycohemoglobin and glycoalbumin are presented in Table 1 and contrasted to human values. There was considerable diversity in the results obtained. The highest glycohemoglobin values were human, the lowest in the chicken and in the pig. It has been demonstrated by other investigators that porcine glycohemoglobin levels are decreased relative to other species due to low erythrocyte glucose permeability in this species (Higgins et al., 1982). In the three avian species studied, the chicken, duck, and turkey, glycohemoglobin levels were similarly very low, despite the very elevated glucose levels in these animals. Conversely, the highest glycoalbumin levels were recorded in these three avian species. This finding is consistent with the direct exposure of albumin to blood glucose while hemoglobin is protected by the erythrocyte membrane. The ratios of glycohemoglobin to glycoalbumin percentages were remarkably different among the animals studied. In an effort to understand the origin of these wide differences further, we analyzed these results with a

[Tot Alb]

kl

percentage

[G] (kl[G] + k21)

(4)

A simple assumption is that glycoalbumin is degraded at the same rate as non-glycosylated albumin. This assumption has actually been verified in the rat (Day et al., 1979). Under this assumption, k2 is obtained from the relationship 0.693 k2 -

t:/2

(5)

where t~/2is the half-life of albumin. Then k, may be obtained from eqn (4) as

(%GA) k, = k2 [O] (1 - (~oOA))"

(6)

An identical series of equations may be derived for glycohemoglobin percentage: d[GHb] dt

k~h[G] [Hb] - kzh[GHb]

(7)

recognizing that k~h is a combined rate constant: klh = k~kp

where k* represents the actual rate constant for formation of glycohemoglobin from glucose and

Table 1. Glycosylated hemoglobin (GlyHb) and glycosylated albumin (GlyAlb) levels in the various species. The number of animals tested (N) is given along with the ratios of the glycosylated hemoglobin and albumin percentages (GlyHb/GlyAlb). Values are given as mean (SE) Animal N Glucose GlyHb GlyAlb GlyHb/GlyAlb 264(6) 88(3) 190(11) 119(5) 103(6) 187(2) 143(19) 142(4) 162(6) 290(7)

(3)

=

RESULTS

7 10 6 7 25 20 4 20 14 4

(2)

The solution of this differential equation is:

(~GA)

Chicken Dog Duck Gerbil Man Mouse Pig Rabbit Rat Turkey

(1)

0.54(0.04) 1.68(0.09) 0.47(0.02) 5.37(0.40) 5.76(0.24) 2.69(0.22) 0.58(0.05) 2.56(0.07) 3.86(0.09) 0.95(0.03)

2.35(0.17) 0.81(0.04) 3.02(0.17) 0.82(0.11) 1.50(0.05) 0.29(0.07) 1.52(0.04) 2.00(0.1l) 0.49(0.05) 3.19(0.21)

0.24(0.03) 2.12(0.16) 0.17(0.01) 7.41(1.16) 3.95(0.09) 11.4(1.4) 0.47(0.04) 1.30(0.05) 10.1(2.5) 0.33(0.06)

Glycosylated Hb and albumin Table 2. Rate constantsfor glycosylatedhemoglobinand albumin. The rate constants for formation of glycohemoglobin(klh) and glyeoalbumin(k~a)are givenin unitsof dl/mg/day.The halflivesof hemoglobinand albuminare indicated,as are the degradationrates k2, and k~ in units/day Hemoglobin Albumin kLh Animal

tt, 2

( × 10-6)

k2h

lu 2

( ×ll0 ) 5)

Chicken Dog Man Mouse Pig Rabbit Rat

8 25 30 16 17 15 15

1.78 5.38 13.6 6.44 1.67 8.44 11.3

0.09 0.028 0.023 0.044 0.041 0.046 0.046

6 8 14 1 8 6 2

1.10 0.78 0.72 1.08 0.91 1.75 1.05

k2 a

0.12 0.085 0.05 0.693 0.085 0.12 0.35

hemoglobin and kp is a constant permeability factor governing the level of glucose in the erythrocyte as a function of plasma glucose concentration. Published half-lives for albumin (Dixon et al., 1953; Kaneko, 1980) and the erythrocytes (Agar and Board, 1983; Archer and Jeffcott, 1979) were available for the chicken, dog, man, mouse, pig, rabbit, and rat. Using these values in the equations, we calculated the rate constants for glycoalbumin and glycohemoglobin in these species (Table 2). This calculation demonstrated a wide variation in values for klh. The lowest values were found in the chicken and the pig, the highest in man and the rat. There was an order of magnitude difference between these values. In contrast, there was far less variation in the calculated values of kta. The lowest value was found in man, the highest value in the rabbit, about a twofold difference.

DISCUSSION By a broad inter-species comparison, we demonstrated a wide diversity in glycohemoglobin and glycoalbumin levels. This diversity is due to species differences in turnover rate of erythrocytes and albumin coupled with differences in rate of formation of the glycosylated proteins. Using a simple mathematical model for the formation of glycohemoglobin and glycoalbumin, it was found that there exists a much greater variability in rate of formation of glycohemoglobin than glycoalbumin. The rate of formation of glycoalbumin from glucose and albumin is quite similar among the species examined. Bunn and coworkers attributed the variable glycohemoglobin rates in dog, pig, man, and ape to marked differences in glucose permeability in the erythrocytes of these species (Higgins et al., 1982). The results reported herein support their work in showing that albumin, which is freely exposed to glucose in the blood, does not exhibit wide differences in species susceptibility to glycosylation. These results suggest that glycoalbumin determinations are a far more useful index of blood sugar levels in species comparison studies than glycohemoglobin. For example, in the avian species, glycohemoglobin levels do not reflect the very high normal blood glucose levels while glycoalbumin levels are highest in the avians. The avian glycoalbumin levels are similar to those found in diabetic human

821

beings (Rendell et al., 1985a,b). It is interesting that normal avians do not develop the common microvascular complications of diabetes. One hypothesis advanced to explain the genesis of microvascular disease is that elevated glucose levels cause increased detrimental glycosylation of certain key proteins in the vascular wall or the basement membrane leading to malfunction. There have been only a few proteins so far identified which are unfavorably affected by glycosylation. Glycosylation of low density lipoprotein markedly alters its interaction with lipoprotein receptors (Gonen et al., 1981; Witztum et al., 1982a,b) thus perhaps relating to the increased atherosclerosis which occurs in diabetic individuals. Glycosylated fibronectin has decreased adhesive properties (Cohen and Ku, 1984). Undoubtedly, many more abnormally functioning glycosylated proteins will be discovered. Why then, if the glycosylated protein hypothesis is true, would the avian species not be affected by microvascular complications? The answer may lie in the low glycohemoglobin levels found in these species. The postulated key proteins may be protected by decreased permeability of the cell membrane to glucose. There needs to be more work on the glycosylated proteins in the various species to identify the key proteins. The comparative approach to the study of the glycosylated proteins may lead to clues helping us to discover the causes of diabetic microvascular complications.

REFERENCES

Agar N. S. and Board P. G. (Eds) (1983) Red Blood Cells o f Domestic Mammals, pp. 80-83. Elsevier, Amsterdam. Archer R. K. and Jeffcott L. B. (1977) Comparative Clinical Haematology, pp. 496-498. Blackwell Scientific, Oxford. Cohen M. P. and Ku L. (1984) Inhibition of fibronectin binding to matrix components by nonenzymatic glyeosylation. Diabetes 33, 970-974. Day J. F., Thornburg R. W., Thorpe S. R. and Baynes J. W. (1979) Nonenzymatic glucosylation of rat albumin. Studies in vitro and in vivo. J. biol. Chem. 254, 9394-9400. Dixon F. J., Maurer P. H. and Deichmiller M. P. (1953) Half-lives of homologous serum albumins in several species. Proc. Soc. exp. Biol. Med. 83, 287-291. Gonen B., Baenziger J., Schonfeld G., Jacobson D. and Farrar P. (1981) Nonenzymatic glycosylation of low density lipoproteins. In vitro effects on cell-interactive properties. Diabetes 30, 875-878. Higgins P. J., Garlick R. L. and Bunn H. F. (1982) Glycosylated hemoglobin in human and animal red cells: role of glucose permeability. Diabetes 31, 743-748. Kaneko J. J. (Ed.) (1980) Clinical Biochemistry of Domestic Animals, pp. 106-107. Academic Press, New York. Klenk D. C., Hermanson G. T., Krohn R. I., Fujimoto K., Mallia A. K., Smith P. K., Wiedmeyer H. M., Little R. R. and Goldstein D. E. (1982) Determination of glycosylated hemoglobin by affinity chromatography: Comparison with colorimetric and ion exchange methods, and effects of common interferences. Clin. Chem. 28, 2088-2094. Mallia A. K., Hermanson G. T., Krohn R. E., Fujimoto E. K. and Smith P. K. (1981) Preparation and use of a boronic acid affinity support for separation and quantitation of glycosylated hemoglobins. Anal. Lett. 14, 645-661. Rendell M., Kao G., Mecherikunnel P., Petersen B., Duhaney R., Nierenberg J., Rasbold K., Klenk D. and Smith P. K. (1985a) The use of aminophenylboronic acid affinity

MARC RENDELL et al.

822

Witztum J. L., Mahoney E. M., Branks M. J., Fisher M., Elam R. and Steinberg D. (1982a) Nonenzymatic glucosylation of low density lipoprotein alters its biologic Rendell M., Kao G., Mecherikunnel P., Petersen B., Duactivity. Diabetes 31, 283-291. haney R., Nierenberg J., Rasbold K., Klenk D. and Smith P. K. (1985b) Aminophenylboronic acid affinity chro- . Witztum J. L., Fisher M., Pietro T., Steinbrecher U. P. and Elam R. L. (1982b) Nonenzymatic glucosylation of high matography compared to thiobarbituric acid technique density lipoprotein accelerates its catabolism in guinea for measurement of glycated albumin. Clin. Chem. 31, pigs. Diabetes 31, 1029-1032. 229-234. Yue D. K., McLennan S., Church D. B. and Turtle J. R. Smith R. J., Koenig R. J., Binnerts A. and Soeldner J. S. (1982) The measurement of glycosylated hemoglobin in (1982) Regulation of hemoglobin A~C formation in human and animals by aminophenyl boronic acid affinity man erythrocytes in vitro: effect of physiologic factors chromatography. Diabetes 31, 701-705. other than glucose. J. clin. Invest. 69, 1164-1168. chromatography

to

measure

J. Lab. Clin. Med. 105, 63-69.

glycosylated

albumin.