Nonenzymatic glycosylation of human plasma low density lipoprotein. Evidence for in vitro and in vivo glucosylation

Nonenzymatic Glycosylation of Human Plasma Low Density Lipoprotein. Evidence for In Vitro and In Vivo Glucosylation Hak-Joong Kim, and Indira V. Kurup To determine the effect of persistent hyperglycemia on the structure of the plasma low density lipoproteins (LDL), nonenzymatic glycosylation of human LDL was studied by incubating LDL isolated from normal subjects with 5 mM or 15 mM glucose in a sterile, buffered solution of pH 7.0 or 9.0, at 4°C. 23°C. or 37°C under nitrogen. The aliquots taken at different intervals showed that the glucose incorporation rate was linear up to the 5th day and was dependent upon pH. The glucose was not incorporated into the lipid portion of the LDL. When the LDL was chemically modified with group specific reagents (diketene for lysyl residues. and cyclohexanedione for arginyl residues), the LDL with modified lysyl residues incorporated glucose much less than untreated LDL, whereas the LDL modified with cyclohexanedione was glycosylated more than the control, suggesting that the lysyl residues are the primary reaction sites. When the amino acids of acid hydrolysates were analyzed by ion exchange chromatography, the radioactivities of either the LDL incubated with glucose and sodium [“H-l borohydride, or the LDL reacted with [U-“C] glucose and sodium borohydride were found in glucosyllysine. The nonenzymatic glucosylation of LDL was also determined with purified LDLs from 6 normal and 6 overtly diabetic subjects. Incorporation of tritium from borohydride, delipidation, acid hydrolysis and chromatography gave radioactivity peaks identified as glycosyllysine. These results suggest that lysyl residues in human plasma LDL can be glycosylated in vivo and in vitro not only in diabetics, but also in normal subjects. The c-amino groups of the lysyl residues on or near the surface of LDL, an important residue controlling its interaction with the LDL receptor(s), are the primary reactive sites. This interaction between plasma glucose and plasma LDL may have relevance in LDL metabolism in diabetic subjects.

A

THEROSCLEROSIS is the leading cause of death in diabetic patients, which frequently occurs prematurely.’ Controversy exists, however, whether hyperglycemia alone is a risk factor for atherosclerosis for this group.‘,’ Although cholesterol-rich plasma low density lipoproteins (LDL) are considered to be a risk factor3 and evidence indicates that prolonged hyperglycemia may be associated with increased plasma LDL-cholesterol,4.S Wilson and Brown, in their review, found no characteristic lipoprotein or lipid abnormality specific for diabetes except for a high incidence of hypertriglyceridemia and hypercholesterolemia;6 the etiology of the increased LDL-cholesterol in diabetes is poorly understood. Bunn and co-workers demonstrated that glucose reacts nonenzymatically with the NH,-terminal resiFrom the Endocrine-Metabolic Section, Department of Medicine. The Medical College of Wisconsin, Milwaukee County Medical Complex, Milwaukee, Wisconsin. Received for publication January 9. 1981. This work was supported in part by the grants from TOPS Club. Inc., Obesity and Metabolic Research program, the American Diabetes Association Wisconsin Afiliate. USPHS General Clinical Research Grant S-MOI-RR 00058. the National Heart, Lung, and Blood Institute Grant HL 27247, and Institutions Research Fund. The Medical College of Wisconsin. This study was presented, in part, at the Vth International Symposium on Atherosclerosis held in Houston. Texas, in November, 1979. and ath the 40th Annual Meeting of the American Diabetes Association, Inc.. held in Washington D.C.. in June 1980. Address reprint requests to Hak-Joong Kim. M.D., EndocrineMetabolic Section, The Medical College of Wisconsin, Milwaukee County Medical Complex. 8700 West Wisconsin Avenue, Milwaukee, Wisconsin 532.26. 0 I982 by Grune & Stratton, Inc. 0026-0495/82/3104~003$01.00/0 348

due of the P-chain (P-NAI valine) to form hemoglobin Ale.’ In addition, glucose forms similar ketoamine linkages with t-amino groups of certain lysyl residues on the CP and P-chains,8.9 and the glycosylation of lysine residues on human hemoglobin is increased in patients with diabetes.8.‘0 This type of post-translational modification has been proposed in the pathogenesis of the long-term complications of diabetes,” and glucose-lysine adducts have been demonstrated in many plasma and tissue proteins including lens crystallin and basic myelin protein.‘2*‘3 Since the lysyland/or arginyl-residues of the plasma LDL are required for its binding to specific receptors, modification of these residues alters their binding to specific receptors and catabolism in vivo.‘4,‘5 We postulated that, if glucose adduction to lysyl residues is increased in diabetic subjects, the catabolism of the modified LDL may be different from the normal, unmodified LDL. The purpose of this study was to examine (1) whether a glucose reaction with the plasma LDL could occur in vitro, (2) if so, whether glycosylation* is at lysinyl residues, and (3) whether similar adduction can be demonstrated in vivo. The results show that glycosylation to lysyl residues of plasma LDL may occur both in vitro and in vivo. MATERIALS

AND

METHODS

Isolation of LDL Human

LDL

(Density

1.02&l ,050 g/ml) was obtained from the

plasma of normal or diabetic

subjects

by sequential

*The term glycosylation was used interchangeably with glucosylation, meaning glucose adduction.

ultracentrifuga-

in this paper

Metabolism, Vol. 3 1, No. 4 (April), 1982

GLYCOSYLATED

tion at 40,000

LDL

rpm in a 50 Ti rotor (Beckman

349

Spinco Division), 4“C

for 20 hr.16 The LDLs were washed twice at density 1,050 g/ml in a SW41TI rotor under the same conditions, and were dialyzed against 61 of 0.15M NaCI, 0.01% EDTA, at 4°C with several changes of the dialysate.

Modification of LDL Reductive methylation of LDL (LDL-protein at 2 mg/ml in 0.15M NaCI, 0.01% EDTA, pH 7.0) was performed at O°C by the addition of 1 mg of sodium borohydride followed by six additions (every 6 min) over 30 min of 1 ~1 of 37% (Wt/Vol) aqueous formaldehyde. (Additions of formaldehyde were made at zero time, 6. 12, 18,24, and 30 min).” The level of modification (% of the total lysine residues) was determined by the trinitrobenzenesulfonic acid (TNBS) calorimetric assay.” Previously, Mahley et al. showed that the calorimetric assay agreed with amino acid analysis.‘s Arginyl groups of LDL were modified by cyclohexanedione.‘8 Lipoprotein (2.5 mg in 1 ml of 0.15M NaClO.Ol% EDTA) was used in 0.2M sodium borate with 2 ml of 0.15M 1,2-cyclohexanedione buffer (pH 8.1) and incubated at 3YC for 2 hr. The LDL was then dialyzed for 16 hr against 0.15M NaCI, 0.01% EDTA at 4°C. This procedure, under the conditions described, consistently resulted in the modification of 50% of the arginine residues of LDL.‘”

Glycosylation LDL (I 5550 mg/ml) solutions were incubated in O.lM phosphate buffer pH 7.4 containing 50 PCi of [U-?Z]-glucose, 5 or 15mM glucose, 0.01% EDTA in pairs. When the higher pH of the solution is desired, 0.1 M borate buffer pH 9.0 substituted the phosphate buffer. Incubations were carried out under N,. at 4”, 22”, or 37°C. After the desired time interval, an aliquot of incubation mixture was taken in duplicate, and was layered on the G-SO column (Sephadex), 9 x 150 mm, which was pre-equilibrated with O.lM phosphate buffer pH 7.4. The eluted protein peak was further dialyzed, 24-28 hr at 4OC against 0.15M NaCI, 0.01% EDTA, pH 7.0 to remove loosely associated glucose radioactivity. Aliquots were assayed for proteinI and radioactivity, and the free amino groups remaining were determined by the TNBS calorimetric assay.” Glucose/protein molar ratio was calculated assuming that the molecular weight of the LDL-apoB is 250.000.t

with 50 gCi of [U-‘“Cl glucose in the presence of 5 mM glucose pH 7.4 for 7 days, and then was reduced with IOO-fold molar excess of unlabeled sodium borohydride as above. Both pairs were dialyzed extensively against 0.15M NaCI, and then against distilled water. The LDLs were delipidated with ethanol-ether.” The lipid fractions were evaporated and the radioactivities were determined as below. The proteins then were hydrolyzed in vacua with boiling HCI for 24 hr. The hydrolysates were evaporated to dryness and stored at 4°C in vacua.

Determination of Glycosyl Residue The identity of the glycosylated residue was determined by chromatography in an ion-exchange column (PA-35 resin, Beckman), 9mm x 150mm. at 32°C. [t4C-] Labeled tyrosine, lysine. histidine, and arginine were used as the marker amino acids. The hydrolysates were dissolved in 0.2M sodium citrate buffer pH 2.5. The residues were centrifuged in a microfuge for I min and the clear supernates were loaded into PA-35 column (9mm x 150mm) and eluted with 0.28M sodium citrate buffer pH 4.5, 70 ml per hr. Fractions of 0.4 ml were collected, and the radioactivities were determined after adding 3 ml of Scintisol Complete (Isolab. Inc.).

RESULTS

When glucose incorporation was expressed as a glucose/protein molar ratio, the glycosylation was linear at 25°C and 37“C both with normal and high glucose concentrations (Fig. 1, 2). The LDL incubated at the higher glucose or temperature was glycosylated twofold more than that incubated at a normal glucose -

0 37°C o 23°C A 4°C

----

Normal

glucose concentration

High glucose concentration

I’

/’

/’

/’

/’

J

/*

Determination of Glucosylation Site Unmodified or modified LDL (1 mg protein /ml) was incubated at 25°C in 0.1 M phosphate buffer pH 7.4 containing I5 mM D-glucose and sterile conditions as described. Duplicate aliquots were removed at intervals to determine the radioactivities and the protein concentration after column chromatography and dialysis. The glucose incorporation after 15 days is expressed as the glucose-LDL protein molar ratio.

Isolation of Glycosylated Residue Poly-L-lysine (Sigma, type I-B) with a molecular weight of approximately 120,000 and the LDL were incubated in two different ways. One pair was incubated with unlabeled glucose (15 mM) at 37OC for 7 days; and then, after the column chromatography and extensive dialysis, the glycosylated proteins were reduced with lOO-fold molar excess of Na [‘HI-borohydride (ICN Pharmaceuticals) (specific activity 12.5 Ci/mol) for one hour at 23OC as described by Bunn and his co-workers.’ The other pair was incubated

tMolecular weight of the human LDL-apoB is not known. Several reports dispute the earlier estimation of 250,000.25

I

DURATION

2

3

4

5

OF INCUBATION(HOURS)

Fig. 1. Comparison of the effects of glucose concentration and temperature on glucose incorporation into human plasma LDL in vitro. Ultracentrifugally purified LDL from normal subjects was incubated in phosphate buffer containing either 5mM (normal glucose) or 15mM (high glucose). 50 j&i [U”C] glucose. Glucose/ LDL-apo molar ratio at different time intervals was determined as described in the Material and Method Section.

350

KIM

l

37°C

-

Normal glucose cmcentraticm High giucose concentration

CONTROL

0

2

3

4

5

6

KURUP

CYCLOHEXANEOIONE REDUCTIVE

I

AND

n.

2

4

6

METHYLATION

8

IO

12

14

DURATION OF INCUBATIONKIAYS) Fig. 2. Comparison of the effects of glucose concentration and temperature on glucose incorporation into human plasma LDL in vitro. Ultracentrifugally purified LDL from normal subjects was incubated in phosphate buffer containing either 5mM (normal glucose) or 16mM (high glucose), 56 lrCi [U”C] glucose. Glucose/ LDL-apo molar ratio at different time intervals was determined as described in the Material and Method Section.

concentration or at 23”C, even after only 5 hr of incubation. Although the higher pH may have had a modest effect on the initial rate of glycosylation, pH did not have any effect when the LDL was incubated at the higher glucose concentration for 2 wk (Table l), suggesting a saturable mechanism at this (glucose) concentration. This was corroborated by the constant number of TNBS reactive group remaining after 10 days of incubations whether at normal or at high glucose concentrations. The modification of arginyl residues increased the glucose incorporation compared to the control (Fig. 3). The LDL modified at the lysyl residue demonstrated Table 1. The Effect of pli on Glycosylation to LDL-apoB AT 37°C’ (Glucose/LDL apo-B molar ratio’) pti of themedia(buffer) Glucose Concentration

7.4

9.0

5

(mM)

15

5

15

Durationof Incubation 5 hr

1.03

2.5

1.2

3.6

5 days

5.4

14.5

4.9

15.3

14 days

6.7

26.5

9.3

25.5

(1.) Only the

data observed at 37°C are shown here. Incubation

conditionswere same as the legendsto Fig. 1 and Fig. 2. (2.) The molecular (3.)

molar weight

Mean

ratio

was

calculated

of the LDL-apoB

of two

separate

was

incubations

under

the

assumption

250,000. in duplicate.

that

the

DAYS Fig. 3. Effects of arginyl- or lysyl-residue modification on glucose incorporation into plasma LDL in vitro. The erginyl residues of normal LDL were pretreated with cyclohexanedione and the lysyl residues were modiied by reductive methylation. Modified LDL (more than 60% of the arginyl or lysyl residues are modified) or intact LDL were incubated in phosphate buffer containing 15mM glucose, 50 PCi [U’k] glucose. Glucose/LDLapoB molar ratio at different time intervals was determined as described in the Material and Method Section.

reduced incorporation of glucose to approximately one-half of that incorporated in control LDL (Fig. 3). Acetoacetylation and acetylation (not shown here) followed similar patterns as shown in the LDL modified by reductive methylation. Since this result suggested that the free amino groups, particularly t-amino groups of the lysine residues may be the site of nonenzymatic glycosylation, we attempted to isolate the residues that are glycosylated. If polylysine is glycosylated in vitro, only glycosyllysine would be the expected product after acid hydrolysis. Both 14C labeled and tritiated polylysine gave identical elution pattern, a double peak (upper panel, Fig. 4). The LDL labeled with 14C or tritium gave similar elution patterns to the polylysine, suggesting that the glycosylated residues are identical to glycosyllysine or its analogue (lower panel Fig. 4). The first major peak was later proved to be a glucosyllysine or its isomer, mannosyllysine, and the second major peak consisted of anhydrous

GLYCOSYLATED

LDL

‘“C-POLYLYSINE

400-

20

40

60

60

100

120

FRACTIONS TYFtOSlNE IZOO-

HISTIDINE

LYSINE

$

+

Fig. 5. Elution profiles of LDL-apoB hydrolysates. LDL isolated from normal or diabetic subjects were treated with sodium [‘HI borohydride without prior incubation with glucose. After delipidation and acid hydrolysis, 200 n moles of the hydrolysates were applied. Poly-L-lysine pretreated with glucose was reacted with sodium TH] borohydride as in Fig. 4, was used as a control.

$

I 1000 -

“C-LDL-apoEl

800 -

diabetic obtained

600-

subjects showed patterns from the LDL glycosylated

similar to those in vitro (Fig. 5).

DISCUSSION

400-

200 -

0 20

40 FRACTION

60

60

I00

120

, 140

NUMBER

4. El&on profiles of protein hydrolysates. One pair of proteins was preincubeted with glucose. and then was treated with sodium CH] borohydride. The other pair was incubated with [U”C] glucose before they were reacted with sodium borohydride. Identical amounts (200 n moles) of amino acids mixtures were applied to the ion-exchange resin, and fractions were analyzed as described in the Material and Method Section.

products of hexosyllysines, an artefact of the acid hydrolysis. A minor radioactivity peak was present immediately after the sample loading (Fig. 4). The identity of the peak is not known at present. To determine wether the LDL is glycosylated in vivo, the LDLs isolated by successive ultracentrifugation from six normal and six overtly diabetic subjects, were reacted directly with tritiated sodium borohydride in the absence of glucose, and were subjected to the same treatment as before (dialysis, acid hydrolysis, and amino acid analysis). The elution patterns of the acid hydrolysates of the LDL from normal as well as

Nonenzymatic glycosylation of proteins, considered in part as browning reaction in the food industry, involves the condensation of carbohydrate and free amino groups of a protein.*‘%‘* The reaction proceeds slowly in the absence of moisture but accelerates when the moisture content increases, or when the materials are in solution.22 However, if the free amino groups are masked, as by acetylation, the reaction does not occur. This was demonstrated in vitro with purified plasma protein (bovine serum albumin) and tissue enzymes (lysozyme and chymotrypsin).** We studied this phenomenon in the relatively short lived plasma protein, plasma LDL. The incubation of LDL with glucose resulted in incorporation of glucose into the LDLapoB. The incorporation is dependent on temperature and glucose concentration of the media (Fig. 1, 2), but is not dependent on incubation pH when the media glucose concentration is high. Since the reaction takes place between the amino groups of protein and the reducible form of glucose, linear incorporation up to the fifth day of incubation and the concentration dependency were expected. Cantor and Pensiston estimated that only 0.04 mole % of total glucose had been present in reducible form in solution in the wide range

352

of glucose concentrations.23.24 The absence of the pH effect, however, was not expected, since the molar percent of the reducible form increases at higher pH values.24 Furthermore, if the primary reaction sites are t-amino groups the glucose incorporation would be expected to be pH dependent, since the pKa of the c-amino group is - 10. More careful study is needed to examine the effect of pH on the glucose incorporation. When more than 50% of the lysyl residues were modified, the glycosylation rate was reduced, but present, suggesting other amino group (a-amino group of N-terminus) may participate in the process (Fig. 3). Recently, several groups of investigators reported phenylalanine as the N-terminus of apoB.25,26 The presence of the glycosyllysine in acid hydrolysate of apoB, but not glycosylphenylalanine strongly suggests that the E-amino group of the lysyl residue is the primary, if not the only, reaction site of the glycosylation, and that either the glycosylphenylalanine may have been destroyed or phenylalanine is not accessible to glucose in the medium. One study supports this possibility.*’ Charge heterogenity, and clustering of basic charges near or at the surface of the lipoprotein,25 a finding which is consistent with the protein modification studies of the lipoprotein controlling its interaction with the LDL receptor,‘4,‘5 are another attractive explanation. Thus, the chemical modification of arginyl residues might enhance the rate of glycosylation of apoB by exposing more lysyl residues near the surface to glucose in the medium (Fig. 3), while a modification of lysyl residues on the surface of LDL-apoB might expose other lysyl residues inaccessible by the reductive methylation. The two peaks of labeled glycosyllysine that appeared on elution profile of acid hydrolysates of both polylysine and apoB (Fig. 4) were expected, since several investigators found this heterogenity in their study of glycosylated hemoglobin and erythrocyte membrane proteins.8*28~29Bailey and co-workers demonstrated that the second peak of the amino acid profile (of the acid hydrosylate) of the glycosylated proteins are anhydrous products of glucosyllysine formed during acid hydrolysis.*’ The identical elution profile in both 3H- or 14C- labeled apo-B indicated the [3H]- borohydride can be used to identify ketoamine linkages present in LDL-apoB. At neutral pH, borohydride has a high degree of specificity for aldehyde and ketone groups (ketoamines in this study), converting them to the corresponding alcohols, although under certain conditions, fatty acid esters can be reduced to extract fatty alcohols.29~30 However, the ethanol-ether of LDL did not contain any significant radioactivities (not shown here). The LDL-apoB that was isolated from normal as

KIM AND KURUP

well as diabetic subjects and subjected to sodium [3H-] borohydride treatment without prior incubation with glucose demonstrated similar elution profiles as the normal LDL incubated with glucose in vitro (Fig. 5), suggesting that glycosylation may occur in vivo in normal as well as diabetic subjects. Our findings add plasma LDL to the growing list of proteins that are glycosylated in vivo. The presence of lysyl residues on the surface of the LDL apparently permit glycosylation even though the half life of the protein is relatively short (3.5 days). Since the LDL receptor-dependent catabolism (binding and internalization) plays an important feedback regulation of cellular cholesterol metabolism and clearance of plasma LDL,” the increased plasma cholesterol in the form of LDL in poorly controlled diabetic subjects4” suggests that hyperglycemia alone may have direct influence on LDL metabolism. Two independent investigations, however, demonstrated that diabetic fibroblasts have no defects in binding and internalization of normal LDL,32,33 suggesting that the presence of glycosylated lysyl residues in LDL-apoB may have relevance to LDL metabolism in diabetic subjects. Preliminary findings in our laboratory suggest that the modified LDL (glycosylated) may have altered catabolism in the cultured human fibroblasts in vitro.34 Since the “rapidly reacting contaminants” are known to be present in the labeled glucose from some commercial sources,35 our data on “C-glucose incorporation to LDL-apoB (Figs. 1 and 2), should be interpreted with a caution. Identical chromatographic characteristics of the glycosyllysines obtained by two difincubation followed ferent methods, i.e., “C-glucose by sodium borohydride, and cold glucose followed by Na [3H]-borohydride seem to suggest that the impurity, if present, was not a significant factor (the top half of Fig. 4). Similarly, when a partially purified 14Cglucose$ incorporation to the LDL-apoB was compared with the glucose incorporation (followed by Na [3H]-borohydride) the incorporation rate was similar (unpublished results). However, a careful study with contaminant free labeled glucose as well as chemically pure unlabeled glucose will be necessary to address this question. ACKNOWLEDGMENT The authors wish to express their thanks to all volunteers, to Drs. Alan Mehler and J. J. Kim of the Department of Biochemistry for their valuable advice on amino acid analysis.

$The rapidly acting contaminants from 14C-glucose was removed by incubating the ‘%-glucose with the polylysine in a dialysis bag or across the dialysis membrane for 24-36 hr at room temperature.

GLYCOSYLATED

LDL

353

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lipoprotein binding to cell surface receptors of fibroblasts following selective modification of arginyl residues in arginine-rich and B apoproteins. J Biol Chem 252:7279-7287, 1977 19. Lowry OH, Rosenbrough NJ, Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275. 1951 20. Scanu AM, Edelstein C: Solubility in aqueous solutions of ethanol of the small molecular weight peptides of the serum very low density and high density lipoproteins. Relevance to the recovery problem during delipidation of serum lipoproteins. Anal Biochem 44576-588. 1971 21. Reynolds TM: Chemistry of nonenzymatic browning II. Adv Food Res 14: 167-283,1965 22. Mohammad A. Frqenkel-Conrat H, Olcott HS: The “browning” reaction of proteins with glucose. Arch Biochem 24:1577178, 1949 23. Pigman W, Anet EFLJ: Mutarotations and actions of acids and bases. in The Carbohydrates. Chemistry and Biochemistry. Pigman W, Horton D, eds. Academic Press, New York, 2nd ed. IA, 1972, pp 165-194 24. Cantor SM, Peniston 1P: The reduction of aldoses at the dropping mercury cathod: estimation of the aldehyde structure in aqueous solutions. J Am Chem Sot 62:2113-2121. 1940 25. Bradley WA, Rohde MF, Gotto Jr AM: Studies on the primary structure of apolipoprotein B. Ann N Y Acad Sci 3488 87-103, 1980 26. Olofsson S-O, Bostrom K, Svanberg U, et al.: Isolation and partial characterization of a polypeptide belonging to apolipoprotein B from low-density lipoproteins of human plasma. Biochemistry 19:1059-1064, 1980 27. Huang SS, Lee DM: A novel method for converting apolipoproteins, into a water soluble protein. Biochem Biophys Acta 5777 424441, 1979 28. Bailey AJ, Robins SD, Tanner MJA: Reducible components in the proteins of human erythrocyte membrane. Biochem Biophys Acta 434:5 I-57, 1976 29. Miller JA, Gravallese E, Bunn HF: Nonenzymatic glycosylation of erythrocyte membrane proteins. Relevance to diabetes, J Clin Invest 65:896-901, 1980 30. Nichols BW, Safford R: Conversion of lipids to fatty alcohols and lysolipids by NaBH,. Chem Phys Lipids 11:222-227. 1973 31. Goldstein JL, Brown MS: Atherosclerosis: the low density lipoprotein receptor hypothesis. Metabolism 26: 1257-l 275, 1977 32. Chait A, Bierman EL, Albers JJ: Low density lipoprotein receptor activity in fibroblasts cultured from diabetic donors. Diabetes 28:914-918, 1979 33. Howard BV, Fields RM, Mott DM, et al: Diabetes and cell growth-lack of difference in growth characleristics of fibroblasts from diabetic and nondiabetic Pima Indians. Diabetes 29: I 19-124, 1980 34. Kim HJ, Kurup IV: Decreased catabolism of nonenzymatitally glycosylated plasma low density lipoprotein by human fibroblasts. Endocrinology 108:273 Abstract No. 764. 198 I 35. Triieb B, Holenstein CG, Fischer R, et al: Nonenzymatic glycosylation of proteins. A warning. J Biol Chem 255:6717-6720, 1980