Acta Diabetol 32: 38-43, 1995 ACTA DIABETOLOGICA (cid:14)9 Springer-Verlag 1995 Non-enzymatic glycation of epidermal proteins of the stratum corneum in diabetic patients I. Mfirovti, J. Zfihejsk#, H. Sehnalovfi Department of Dermatology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Received: 21 January 1994/Accepted in revised form: 18 October 1994 Abstract. A selected group of diabetic patients showed a statistically significant increase in levels of glycated proteins in the stratum corneum compared with a control group. The values of glycated proteins correlated with those of glycohaemoglobin (GHb), and in diabetic pa- tients also with serum glucose concentrations. The values of glycated proteins (and GHb) exhibited a positive cor- relation with age both in a control group and in diabetic patients. The average values of glycated proteins (and GHb) were slightly higher in women than in men. Deter- mination of glycated proteins levels of the stratum corneum can serve as a stable parameter for long-term monitoring of the course of non-enzymatic gtycation in structural and connective tissues and thus also for the prognosis of the development of dermatological compli- cations related to diabetes mellitus. In vitro incubation of stratum corneum proteins and keratin with glucose re- suited in an increase of their glycation. The values of glycated proteins and glycated keratin increased propor- tionally to the glucose concentration and duration of in- cubation. Glucose binding to keratin and proteins of the insoluble stratum corneum fraction appeared to occur at practically the same rate, and it is a first-order reaction with regard to the glucose concentration. Water-soluble proteins of the stratum corneum undergo non-enzymatic glycation preferentially (on average, 83.4% of the total amount of glycated proteins is present in the soluble frac- tion), regardless of the initial content of glycated proteins in the sample. The content of glycated soluble proteins of a higher molecular weight significantly increased after 4 weeks of incubation with glucose. Key words: Non-enzymatic glycation - Epidermal proteins - Stratum corneum - Diabetes mellitus Correspondence to: J. Z~ihejsk~, Department of Dermatology, Peka~skfi 53, 656 91 Brno, Czech Republic Introduction Non-enzymatic glycation of proteins occurs in vivo after long-term exposure of proteins to higher glucose concen- trations. The essence of this modification process is a chemical reaction between a carbonyl group of glucose and a free amino group of the protein, generally called the Maillard reaction [1]. In connection with the pathogenesis of diabetes, the above-mentioned modification reaction was first report- ed when glycohaemoglobin (GHb) was discovered [2]. Increased non-enzymatically glycated Hb concentrations in diabetes mellitus [3, 4,] led to intensive research into similar excess glycation of other tissue proteins, especially in an attempt to establish a link between this process and the chronic complications of diabetes mellitus. Since then, the non-enzymatic glycation of a number of proteins from all tissues of the human organism has been demonstrated in healthy subjects and particularly in dia- betic patients [5]. At present, non-enzymatic glycation seems to be only the first step of a complex sequence of Maillard reactions, referred to in ageing individuals as "non-enzymatic browning" [6]. In time, the Amadori products of glycation gradually undergo a series of sequential reactions and changes resulting in the formation of"advanced glycation endproducts" (AGE) [7]. These changes include protein denaturation and polymeration, formation of rigid cross- linked structures and generation of specific protein- bound fluorophores. These adducts are associated most often with long-lived proteins such as lens crystallin, col- lagen and myelin and have also been detected in vivo [6-111 . It has been shown that keratin proteins also undergo non-enzymatic glycation [12-14]. The aim of the present work is to follow the glycation of keratin and other proteins of the stratum corneum, and thus contribute to the understanding of the course of modification reactions in tissues, their consequences and potential relation to the development of skin disorders in dermatological patients with diabetes.
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`I. Mfirov/t et al.: Non-enzymatic glycation of stratum corneum epidermal proteins Materials and methods The study group included 148 diabetic patients being treated at the 1st Department of Dermatovenerology in Brno for various diseases, mainly leg ulcerations and microbial eczema. The group comprised 78 women and 70 men aged 19-85 years. Patients were divided into four groups according to the character of their diabetes: (i) type I (DM I), (2) type II (DM II), (3) first diagnosed during hospitaliza- tion (DM n) and (4) impaired glucose tolerance (IGT). A group of 50 patients (25 men and 25 women) who evidently did not suffer from a saccharide metabolism disorder was used as the control group. The stratum corneum specimens were taken from the sole sur- face area, without applying any external therapy, using a dermato- logical shaver. On average, a specimen of 300 mg stratum corneum was taken from each patient. The specimen was dried to constant weight (2 h, 60 ~ and homogenized (Retsch). Weight losses related to drying were within 7%-13% of the initial specimen weight (9.5% on average). Simultaneous with the stratum corneum specimen, a blood sam- ple was taken for the determination of GHb. 39 in 0.05 tool/1 TRIS-HC1 buffer, pH 8.6, with 0.02% NaN 3. The flow rate was adjusted at 25 ml/h. In the 2.5-mt fractions the protein concentration was determined, and the presence of GProt was de- tected colorimetrically. Affinity chromatography was used only for the determination of soluble GProt. Characterisation of proteins Gelfihration. A sample of soluble GProt (usually 175-200 mg dry weight in 3.5 ml buffer, i.e. approximately 40-60 mg protein in 0.01 tool/1 phosphate buffer, pH 7.4) was applied to the column K 16/40 (Pharmacia) filled with about 70 ml Sephadex G-15 and equilibrated with 20-30 volume units of the same buffer. Separa- tion continued at a constant flow rate of 0.4 ml/min. The eluate was collected into 2-ml fractions. Free amino groups determination. Separated soluble GProt from the stratum corneum extract and also joint fractions from gel filtration were examined for free amino groups. Samples were treated with 2.4-dinitrofluorobenzene, and groups identified by thin-layer chro- matography [18]. Assays The serum glucose concentration was determined by the enzyme photometric method using the Oxochrom glucose diagnostic kit (Lachema). Hb was determined colorimetrically using the glycosy- lated haemoglobin diagnostic kit (Lachema). To analyse protein concentration in the stratum corneum and in the fractions obtained by chromatography, the colorimetric method according to Lowry et al. [15] or the measurement of absorbance at 280 nm was used. Electrophoresis PAGE-SDS. The joint lyophilized fractions from gel filtration were used as samples. The optimized SDS-polyacrylamide gel electrophoresis [19, 20] using a gradient of N,N'-methylene- bisacrylamide (0.5%-3%) was carried out in a protein double slab electrophoresis cell (Bio-Rad). Electrophoresis conditions were: gel gradient of 8%-21%, gel surface of 0.15 x 14 cm, separation time of 6.5 h at constant current of 25 mA. LMW calibration kit (Pharmacia) was used as the molecular weight standard. The protein zones were stained with Coomassie blue R-250. The electropherograms were analysed in a Vitatron densitometer. Measurement of non-enzymatic glycation Sample preparation. For the usual determination of total glycated proteins of the stratum corneum (GProt), a sample of 50 mg dried homogenized preparation of the stratum corneum was suspended in 1.5 ml solvent (distilled water or buffer if the sample was chro- matographed: gel filtration, 0.01 tool/1 phosphate buffer, pH 7.4; affinity chromatography, 0.05 mol/l TRIS-HC1 buffer, pH 8.1). Be- fore separation of the soluble fraction, the stratum corneum speci- men was suspended in a solvent (see above) for a period of 1 h while shaken intermittently and then centrifuged. The soluble fraction (supernatant) was used directly for analysis while the insoluble fraction was washed several times in a solvent. When determining the total GProt or pure keratin, the assay was performed directly after suspending. Colorimetric determination of glycated proteins. Our own modi- fication of a colorimetric method [16] based on the detection of a yellow product generated by a reaction of 5-hydroxymethylfurfural (5-HMF) and thiobarbituric acid [I7] was used for the determination. Treated samples were hydrolysed with 85% phosphoric acid (1 h, 100 ~ After cooling, ballast proteins were separated from the sample by precipitation with cool trichloracetic acid (2.45 tool/l), followed by centrifugation. The supernatant was then used for the colour reaction with thiobarbituric acid (2.5 tool/l) in a ration of 2:1. After 40 rain at 30~ in a water bath, the colour intensity was determined photometrically at 443 nm (Cary 118 spectrophotome- ter; Varian). Each series analyzed contained a control sample of the standard GProt value, fructose calibration solutions (0.1-0.3 mmol/1) and a blank. The amount of GProt was expressed in gmol of fruc- tose per gram of dry weight. Affinity chromatography. A column filled approximately with 10 ml boronic acid agarose gel was equilibrated with 20-30 volume units of a starting buffer (0.05 tool/1 TRIS-HC1, pH 8.1, with 0.02% NAN3). Elution of GProt was accomplished using 0.3 mol/1 sorbitol In vitro glycation A simple model systems for studying the non-enzymatic glycation of pure keratin and a mixed preparation of the stratum corneum homogenate of an initial value of total GProt = 3.2 pmol F/g dry weight was used. Keratin or stratum corneum (50 rag) was incubated in a suspen- sion of 0.01 mol/1 phosphate buffer, pH 7.4, containing antibiotics and NaN 3 with glucose in a concentration range of 0-30 mmol/1 at 10 ~ In samples the initial value of GProt and the values after 1, 2, 3, 4 and 8 weeks of incubation were determined. Free glucose was removed from the incubation mixture for keratin and insoluble GProt by washing it several times with distilled water before the determination. The determination of GProt or glycated keratin was performed colorimetrically. From separated soluble GProt, free glucose was removed by ultrafiltration using the membrane filter YM 05 (Amicon). The soluble GProt level was determined using both a colorimetric method and affinity chromatography as an orientation method. A chromatographic analysis of soluble GProt at 1-week intervals of incubation was done by gel filtration. Statistical methods The values of GHb, glycated proteins and glucose in diabetic pa- tients and a control group were compared using the t-test. Correla- tions between the values of individual parameters were calculated using the Spearman correlation coefficient. Chemicals Purified keratin from cornea, insoluble in water, was purchased from Merck. Boronic acid agarose was the product of ICN Biomed- icals (UK). Sephadex G-15 as well as LMW calibration kit L 14
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`40 I. M/trovfi et al.: Non-enzymatic glycation of stratum corneum epidermal proteins (4000-94 000) were from Pharmacia (Sweden). Other materials for electrophoresis separation were mostly from Serva, Heidelberg (Germany). Membrane filter YM 05 was obtained from Amicon (USA). Results Evaluation of a group of patients Table 1 presents the mean values of GHb, glycated stra- tum corneum proteins (total) and serum glucose concen- trations in a control group and in individual groups of diabetics. The highest mean values of GProt were observed in patients with recently discovered and so far non-compen- sated diabetes. A normal range of GProt values was de- termined on the basis of evaluating the results of a con- trol group as 1.5-3.2 ~tmol F/g dw. Within the group of patients evaluated, a positive cor- relation was found between the values of GHb and total GProt (at a significance level of 1%), particularly in dia- betic patients (r=0.682) and in a control group (r=0.579). In a group of diabetic patients, correlation was also found between the values of GProt and serum glucose (r = 0.545). Table 1. Glycohaemoglobin, glycated proteins of the stratum corneum and serum glucose measurements Group n Glyco- Glycated Glucose haemoglobin proteins SD ~ SD ~ SD Control 50 4.9 0.6 2.5 0.5 4.7 0.5 DM I 12 9.3 1.4 3.9 0.8 12.6 6.6 DM II 98 7.8 1.3 3.6 1.3 10.1 2.8 DM n 25 8.4 2.7 4.3 1.5 12.0 4.9 IGT 13 5.2 0.4 2.8 0.7 6.6 1.1 DM I, Type I diabetes mellitus; DM II, type II diabetes mellitus; DM n, diabetes mellitus first diagnosed during hospitalization; IGT, impaired glucose tolerance [pathological OGT (T)] Table 2. Correlation between protein glycation and patient's age and sex Group n GHb GProt 2 SD r 2 SD r Women 103 Control 25 5.18 0.73 0.709 2.78 0.87 0.690 Diabetic 78 8.37 1.16 0.710 3.76 1.09 0.569 Men 95 Control 25 5.11 1.21 0.708 2.65 0.56 0.930 Diabetic 70 7.25 0.98 0.630 3.68 1.21 0.763 Whole group 198 Control 50 5.15 0.68 0.889 2.62 0.67 0.872 Diabetic 148 7.81 1.54 0.662 3.77 1.14 0.628 r, Correlation coefficient (with age) GProt I ,umol F I g d w / (cid:12)9 (cid:12)9 ii (cid:12)9 (cid:12)9 (cid:12)9 n uno u~176 (cid:12)9 (cid:12)9 (cid:12)9 ~149149149176 (cid:12)9 (cid:12)9 (cid:12)9 o ." ".," ." ", .o: (cid:12)9 (cid:12)9 ", (cid:12)9 _% o,.o ..,. ,%oo.O.~.o_~. 3-t (cid:12)9 "" [] [] .... ~.o,, o~,~soo'~ .." (cid:12)9 BOg D ~ OO --I oD oOO ~ o ~~176176 "(cid:12)9 (cid:12)9 2- nu De [] muua~~ aoD o u mUo 1 [ ,ore , , , 30 40 50 60 70 80 Age Fig. 1. Glycated proteins (GProt) versus age: (cid:12)9 female diabetics; (cid:12)9 male diabetics; o female control group; [] male control group The dependence of GHb and GProt levels on age was followed both in the whole group of diabetic patients and in a control group, and in groups of men and women control and diabetic patients. The results obtained in- cluding the correlation coefficients (evaluated at a signif- icance level of 1%) are summarized in Table 2. Figure 1 illustrates graphically the dependence of GProt values on the patients age. Slight differences in GHb and GProt levels were ob- served between women and men in the control group, while the differences between these values were somewhat higher in diabetics. In both groups (male and female) as well as in the whole group of diabetic patients and also in a control group, a good correlation was observed between GHb and GProt values and the subjects' age (see Table 2). Characterisation of glycated proteins Distribution between soluble and insoluble fractions. The amount of total GProt was determined in 12 selected specimens of the stratum corneum. The soluble fraction contains on average 83.4% of the total amount of GProt regardless of whether the specimen came from a healthy individual or a diabetic patient with a higher level of protein glycation. Gel filtration. High-porous (Sephadex G-100) and low- porous (Sephadex G-50, G-25, G-15 and G-10) gels were tested in preliminary experiments. Sephadex G-15, en- abling the most efficient course of separation of predom- inantly low-molecular weight (LMW) proteins of the tis- sue examined, was selected for the chromatographic anal- ysis. Figure 2 compares the elution profiles of the samples with normal and pathological initial values of GProt in a series of samples with increasing GProt values. From the chromatograms it is evident that glycated proteins were separated into three larger and two smaller groups ac- cording to their molecular weight. Maximum IV and V are detectable only in samples with higher total GProt
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`I. MArovA et al.: Non-enzymatic glycation of stratum corneum epidermal proteins 41 A 1.0- 0.8- 0.6- 0.4- 9.2-1 0! t ft | /!",,.\ ~ \ l //%'.'., - \ / ,s ~.z,.....? ...... :~---~).~_. ,/~'....-.-...<~.~."''>.':'-"-~S'"" "" ':.'."" "':.:.:7."7. i 10 20 30 V /, ,j \ . 40 m Ve , ml Fig. 2. Chromatography of samples with an increasing content of total GProt using Sephadex G-I 5. Samples: 30-50 mg of proteins, raw extract applied. Ve, Elution volume; solid line, profile of protein concentration in the eluate; a, b, c, d, glycated proteins in fractions; GProttotal=2.2 (a), 2.8 (b), 4.5 (e) and 6.0 (d) gmol F/g dw; I-V, groups of glycated proteins with gradually decreasing molecular weight. Note: Profiles of protein concentration for individual sam- ples are similar. Therefore, only one shown for illustration (each sample is from another patient) Table 3. Determination of free amino groups in soluble fraction of the stratum corneum extract Sample Before hydrolysis After hydrolysis (gm91 -NH2/mg dw) (I-tmol -NHz/mg dw) Crude extract 5.50 2.03 Soluble GProt Fraction I 1.41 0.57 II 0.94 0.65 III 0.94 0.33 IV + V 4.24 0.27 values. Proteins of the IInd maximum are glycated preferably. The increase of the peak area is linear with the increasing initial value of total GProt. Free amino groups determination. Results are summa- rized in Table 3. The number of fractions corresponds to the number of fractions from gel filtration (see Fig. 2). The amount of free amino groups in samples analysed both before hydrolysis and afterwards corresponds to the previously described composition of stratum corneum extracts [21]. Molucular weight determination. PAGE-SDS electro- phoresis was used for more exact determination of the molecular weight in individual protein groups obtained by gel filtration. Figure 3 shows the results, including separation of the set of LMW standards. We managed Fig. 3. PAGE-SDS electrophoresis: L II, III, Groups of proteins in the same order in which they are eluated from Sephadex G-15; LMW, calibration set of standards of low molecular weight to detect only proteins related to the first two peaks (see Fig. 2), where the Ist maximum is related to a larger number of various proteins of molecular weight ranging from 10000 to 70000 Da. Only three clear bands corre- sponding to proteins of presumed molecular weight of 13 000-15 000 Da are associate with maximum II. In vitro glycation Figure 4 presents changes of the values of glycated proteins or keratin and their dependence on the duration of incubation in a medium with increasing glucose con- centrations (within physiological range). From Fig. 4 it is evident that the values of GProt and glycated keratin increase proportionally with glucose concentration and the duration of incubation. The rate of glycation was practically indentical in both cases. Glucose binding to keratin and water-insoluble proteins of the stratum corneum is a first-order reaction with regard to glucose concentration. The value of insoluble GProt represents, on average, 16.7% of the total value of GProt regardless of the length of incubation. In addition to a colorimetric method, the control de- termination of the soluble GProt fraction was done by affinity chromatography using boronic acid agarose. The GProt level was also determined in fractions colorimetri- cally, while evaluation of the total GProt (%) by affinity chromatography was done only from the values of AE80 in fractions. From the first experiments it is evident that the values of soluble GProt measured colorimetrically increase somewhat faster than those obtained by affinity chromatography.
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`42 I. M&rovfi et al.: Non-enzymatic glycation of stratum corneum epidermal proteins G~I Keratin z~ l [glu] 0.5-I (cid:12)9 o 0 ~ (cid:12)9 .... '/ 0 1 2 3 4 Weeks 8 GProt pmol F 0 01 0 [] S. corneum ~ z~ II0,ul A O O [] O // i 3 4 Weeks ; Fig. 4. Dependence of the value of glycated proteins and glycated keratin on the duration of incubation in various glucose concentra- tions in an incubation medium in vitro. Glucose concentration in medium: 0, 5 (o), 10 (o), 15 (x), 20 (zx) and 30 (rT) mmol/1 From the results of chromatographic analysis of soluble GProt after in vitro incubation with glucose, it is clear that after 4 weeks of incubation the content of GProt of higher molecular weight increased significantly (chromatograms not shown), with a corresponding de- crease of LMW GProt. In all the groups followed, the portion of glycated proteins increased proportionally with the duration of incubation. Discussion As a result of post-synthetic modifications of the non- enzymatic glycation type, significant changes of physical and functional properties of proteins occur. Glycation of connective tissue proteins results, in addition to reduced solubility and higher thermal stability, in a higher tenden- cy to form intramolecular cross-linkages between protein filaments, as was described for collagen [11, 22]. In the case of lens crystallin, large aggregates of extremely high molecular weight are formed [23]. A possible correlation between higher glycation, cross-linking and the incidence of neuropathic ulceration was described for keratin [12]. In fact, for various skin disorders, a higher rigidity of tissue structures was observed in diabetics patients [24, 25]. The major protein component of the stratum corneum is keratin, a fibrous scleroprotein characterized by high insolubility and resistance to enzymic lysis and autolysis. In addition, the stratum corneum contains 26.3 % of wa- ter-soluble substances of which protein make up 2.4% [21]. Based on our results it is possible to conclude that the conditions for glycation in the stratum corneum are better met by soluble proteins, when compared with in- soluble ones. On the other hand, irreversible modifica- tion of a keratin molecule results in more serious physio- logical consequences due to its long lifetime and structur- al and mechanical functions. According to data in the literature, glycated proteins are more reactive than non- glycated proteins ( e.g. IgG binding to glycated collagen is 4 times faster than binding to non-glycated protein; [26]). Consequently, we assume that a high proportion of soluble GProt could function in the process of glycation in the stratum corneum as an available reactive potential, becoming involved in binding to keratin in late phases. This problem deserves further investigation. From the literature dealing with glycation of hair and nail keratin [13, 14] it is evident that the level of glycated keratin can serve as a stable parameter for long-term monitoring of glycaemia and its compensation. The com- parison of data on hair glycation [13] and our results obtained in a double group of patients suggests that gly- cation of the stratum corneum keratin can also be used for this purpose. However, we assume that use of this parameter would be more valuable to estimate the degree of glycation in tissues, and thus to predict complications related to diabetes. In dermatology, this parameter can be used to estimate the course of a disease and its healing capacity and can also serve as auxiliary data when select- ing therapy. While evaluating our patients, we found slightly higher values of GHb in women than in men, both in a control group and in diabetic patients (see Table 2). Greater differences were observed in GProt values, which were also higher in women. For the diabetic patients, neither the duration of the disease nor the kind of therapy were taken into consideration during the evaluation; however, most of them were taking insulin. We assume that the differences have practically no significance with regard to the disease. Similar data can also be found in the literature, even though the sex difference has not been explained [27]. Within our group of patients, there was a relatively good correlation between the levels of glycated proteins and GHb and age (Table 2). While the GHb concentration should not causally depend on age, a similar correlation was described in healthy people and in diabetic patients [28, 29]. In the case of GProt, a dependence on age is more likely and can be explained analogously to the reactions occurring during glycation and tissue ageing [11, 30]. Methodological approaches to the monitoring of gly- cation of the stratum corneum proteins are limited by the fact that the sample contains water-soluble and -insolu- ble fractions. To assess any sample containing an insolu- ble fraction, it is only possible to use a colorimetric meth- od. This is universally applied, furthermore, its results are not influenced by a labile form. Soluble GProt can also be assessed by other methods, of which affinity chro- matography is the best for the sample composition
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`I. Mfirov~ et al.: Non-enzymatic glycation of stratum corneum epidermal proteins 43 [30, 31]. The repeatedly confirmed fact that a portion of GProt is not bound by the affinity gel under the conditions applied (see Methods) requires further investigation. A major proportion of soluble GProt has a relatively low molecular weight, which is in agreement with data in the literature as well as the results of PAGE-SDS analysis (see Fig. 3). Proteins found in the stratum corneum were pre- dominantly of 5- 30 kDa with a maximum of 69 kDa [21]. The extent of glycation of keratin and proteins in a complex sample of the stratum corneum observed in vitro increased proportionally to the glucose concentration and duration of incubation (Fig. 4). The duration of incu- bation had no influence on protein distribution between the soluble and insoluble fractions. From comparison of the extent of glycation of keratin and an insoluble fraction of the stratum corneum extract, it is evident that the rate of glycation is practically identical in both cases. This re- sult is in agreement with the data on the composition of the stratum corneum, half of which consists of keratin. It is probably a reaction of the same protein with glucose at the same binding site. Glucose binding to keratin and insolu- ble stratum corneum proteins is a first-order reaction with regard to glucose concentration, suggesting the presence of a single, rapidly modified amino acid residue and other less reactive secondary sites of the protein molecule. Further experiments are necessary to verify this assumption. A significant increase of GProt of higher molecular weight after 4 weeks of incubation probably means that oligomers are formed, consisting of LMW proteins or other types of aggregates. Similar reactions were ob- served after an 8-day incubation of albumin in 0.2 tool/1 glucose [32], during which albumin dimers to hexamers were formed. Similar processes could occur also during a reaction of soluble proteins and keratin. The results presented suggest that glycation of the stratum corneum proteins is subject to certain laws appli- cable to proteins of other tissues, although many of them remain to be explained. Keratin glycation probably plays a role in the development of dermal complications related to diabetes even though the evidence of a direct connec- tion does not exist. In this respect we believe that, in addition to the monitoring of glycation processes and their causes, the study of the possibilities of targetting the course of glycation and thus the prevention of decelera- tion of tissue damage will be of great significance. References 1. Maillard LC, Reaction generale des acides amines sur le sucres: ses consequences biologiques. CR Acad Sci 154: 66-68, 1912 2. Rahbar S, An abnormal haemoglobin in red cells of diabetics. Clin Chim Acta 22:296-298, 1968 3. Gabbay KH, Sosenko JS, Banuch GA, Minisohn M J, Fliickiger R, Glycosylated haemoglobins: increased glycosylation of haemoglobin A in diabetic patients. Diabetes 28: 337-340, 1979 4. Bunn HF, Gabbay KH, Gallop PM, The glycosylation of haemo- globin: relevance to diabetes mellitus. Science 200:21-27, 1978 5. R/tcz O, Vicha T, Pa6in J, Glycohaemoglobin, glycation of proteins and diabetes mellitus. Osveta Martin (SR), 1989 6. Monnier VM, Kohn RR, Cerami A, Accelerated age-related browning of collagen in diabetes mellitus. Proc Natl Acad Sci USA 81:583-587, 1984 7. Brownlee M, Cerami A, Vlassara H, Advanced glycosylation end-products in tissue and the biochemical basis of diabetic complications. N Engl J Med 318: 1315-1321, 1988 8. Monnier VM, Stevens VJ, Cerami A, Nonenzymatic glycosyla- tion, sulfhydryloxidation and aggregation of lens proteins in experimental sugar cataracts. J Exp Med 150: 1098-1107, 1979 9. Day JF, Thorpe SR, Bayers JW, Nonenzymatically glucosylated albumin. J Biol Chem 254: 595-597, 1979 10. Schnider SL, Kohn RR, Effect of age and diabetes mellitus on the solubility and non-enzymatic glycosylation of human skin collagen. J Clin Invest 67:1630-1635, 1981 11. Schnider SL, Kohn RR, Glycosylation of human collagen in aging and diabetes mellitus. J Clin Invest 66:1179-1181, 1980 12. Delbridge L, Ellis CS, Robertson K, Lequesne LP, Non-enzy- matic glycosylation of keratin of the stratum corneum of the diabetic foot. Br J Dermatol 112: 547-554, 1985 13. Paisey RB, Clamp JR, Kent MJC, Light ND, M, Hartog M, Glycosylation of hair: possible measure of chronic hypergly- caemia. Br Med J 288: 669-671, 1984 14. Bakan E, Bakan N, Glycosylation of nail in diabetics: possible marker of long-term hyperglycaemia. Clin Chim Acta 147: i- 5, 1985 15. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ, Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265-275, 1951 16. Sehnalovfi H, Z/thejsk~ J, Photometric determination of glyco- sylated epidermal keratin. Biochem Clin Bohemoslov 19: 351- 358, 1990 17. F1/ickiger R, Winterhalter KH, In vitro synthesis of haemo- globin Ale. FEBS Lett 71: 356-360, 1976 18. Ghusyen JM, Tipper DJ, Strominger JL, Enzymes that degrade bacterial celt walls. Methods Enzymol 8: 695, 1966 19. Racek P, The optimized SDS-polyacrylamide gel electrophore- sis using a gradient of N,N-methylene-bis-acrylamide. Bull Czech Biol Soc 11:101, 1983 20. Laemmli UK, Cleavage of structural proteins during the assem- bly of the head of bacteriophage T 4. Nature 227: 680-683, 1970 21. Hook B, Neufahrt A, Leonhardi C, Separation of water proteins in psoriatic scales with different polyacrylamide gel concentra- tions and molecular weight estimations of the separated bands by disc electrophoresis. Arch Dermatol Forsch 250: 245-252, 1974 22. Yamauchi M, Woodley DT, Mechanic GL, Aging and cross-link- ing of skin collagen. Biochem Biophys Res Commun 152:898-903 23. Kasai K, Nakamura T, Kan N, Suzuki R, Fogure R, Shimoda S, Increased glycosylation of proteins from cataractous lenses in diabetes. Diabetologia 25: 36, 1983 24. H6dl S, Skin disorders in diabetes mellitus. Acta Derm Venerol 1:71-76, 1992 25. Vishwanath V, Frank KE, Elmets CA, Dauchot P J, Monnier VM, Glycation of skin collagen in type I diabetes mellitus. Corre- lation with long term complications. Diabetes 35: 916-921, 1986 26. Brownlee M, Pongor S, Cerami A, Covalent attachment of soluble proteins by nonenzymatically glycosylated collagen. J Exp Med 158:1739-1744, 1983 27. Stickland MH, Paton RC, Wales JK, Haemoglobin Ale concen- trations in men and women with diabetes. Br Med J 289: 733- 739, 1984 28. Oimoni M, Masumoto S, Hatanaka H, Hemoglobin A 1 and hemoglobin Ate in elderly diabetes. Kobe J Med Sci 31:95-101, 1985 29. Graf JR, Halter JB, Porte D, Glycosylated hemoglobin in nor- mal subjects and subjects with maturity-onset diabetes. Dia- betes 27: 843-839, 1978 30. Monnier VM, Cerami A, Non-enzymatic browning in vivo: possible process for aging of long-lived proteins. Science 211:491-493, 1981 31. YascoffRW, Tevaarwerk GJM, MacDonald JC, Quantification of nonenzymatically glycated albumin and total serum protein by affinity chromatography. Clin Chem 30: 446-449, 1984 32. Day JF, Thornburg RW, Thorpe SR, Bayness JW, Nonenzymatic glycation of rat albumin. J Biol Chem 254:9394-9400, 1979
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`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00171
`
`