Glycosylation

POS-003-091 ROLE OF GLYCOSYLATION OF PROTEINS IN VARIOUS LONG-TERN COMPLICATIONS OF DIABETES MELLITUS

N. ATAUR RAHNAN, GHAZALA ZAFAR & A. SANAD SHERA*, Department o f B i o c h e m i s t r y , 3 innah Pos tg radua te Med ica l C e n t r e , K a r a c h i , & * D i a b e t i c A s s o c i a t i o n o f P a k i s t a n ,

V / E - 5 Mohd. S idd ique Khan Trus t B l d g . , N. Nazimabad, K a r a c h i , (PAKISTAN).

Changes i n g l y c o p r o t e i n c o n c e n t r a t i o n , i t s compos i t i on and inc reased non-enzymat ic g l y c o s y l a t i o n o f v a r i o u s pro te ins i n d i a b e t i c p a t i e n t s have been repor ted by many worke rs . Present study desc r i bes a c o r r e l a t i o n o f serum g lycop ro te in l e v e l and ex ten t o f non-enzymat ic g l y c o s y l a t i o n w i t h the d i a b e t i c s t a t e in the presence o f c l i n i c a l l y man i fes ted ch ron i c d i a b e t i c c o m p l i c a t i o n s . E igh ty f i v e d i a b e t i c p a t i e n t s were s t u d i e d , out o f these 20 were w i t h o u t any c l i n i c a l ev idence o f c h r o n i c c o m p l i c a t i o n s w h l l e remain ing were s u f f e r i n g from c a t a r a c t (n=18) , r e t i n o p a t h y (n=16) , p e r i p h e r a l neuropathy (n=16) and c a r d i o v a s c u l a r compl ica t ions (n=15) . Fas t i ng b lood sample was c o l l e c t e d from the p a t i e n t s and c o n t r o l s u b j e c t s . Both enzymat ic and non-enzymat ic g l y c o s y l a t i o n o f p r o t e i n s were e l eva ted in a l l d i a b e t i c p a t i e n t s . The increase i n plasma g lucose was c o r r e l a t e d s i g n i f i c a n t l y w i t h g l y c o s y l a t e d haemoglobin, g l y c o s y l a t e d plasma p r o t e i n and serum f ruc tosamine c o n c e n t r a t i o n s . There was no s i g n i f i c a n t d i f f e r e n c e in f a s t i n g plasma g lucose , g l y c o ~ y l a t e d plasma proteins, glycosylated haemoglobin, serum fructosamine, mucoprotein, hexosamine, sialic acid and fucose in diabetic patients with and without chronic complications. Serum proteins were separated electrophretically on cellulose acetate membrane and stained for protein with ponceau stain and for serum glycoproteins with periodic Schiff's reagent. Alpha-2 globulin fraction was increased in both uncomplicated and complicated diabetic patients. In patients with retinopathy beta-globulin was decreased, alpha-2-glycoprotein was elevated significantly and beta-glyco- protein was decreased. In uncomplicated diabetic patients alpha-l-glycoprotein was decreased and

gamma-glycoprotein was significantly elevated.

POS-003-092 THE EFFECTS OF GLYCOSYLATED SERUM PROTEINS ON PROSTAGLANDIN SYNTHESIS IN PLATELETS AND CULTURED ENDOTHELIAL CELLS

H KISHIKAWA, H TAKEDA, M SHINOHARA, H MATSUDA, M SHICHIRI Department of Metabolic Medicine, Kumamoto university Medical school, Kumamoto, Japan

The effects of native or glycosylated serum proteins on arachidonic acid-induced prosta- glandin production in platelets and cultured endothelial cells in vitro were evaluated. Washed platelet suspensions from 8 type 2 diabetic subjects were prepared from the EDTA- treated platelet rich plasma by centrifugation, which were finally suspended in Tris- saline-dextrose buffer (PH 7.4). Human umbilical vein endothelial cells were cultured according to a method by Jaffe et al, with slight modification. The endothelial cells were used in passages 2-4. Low density lipoproteins (LDL) were prepared by ultracentrifugation. LDL and human serum albumin (HSA) were glycosylated by incubating with glucose (80mM) and NaCNBH 3 (200mM). The platelet suspensions or confluent endothelial monolayer cells were incubated with native or glycosylated HSA (1.5 mg prot./ml), LDL (0.2 mg prot./ml), or physiological saline for i0 minutes at 37°C. They were stimulated by arachidonic acid (400PM for platelets, 20PM for endothelial cells) for 5 minutes at 37°C. Thromboxane B 2 or 6-keto PGFIa content of the supernatant was determined by radioimmunoassay. Native LDL caused a greater increase in arachidonio acid-induced platelet thromboxane B 2 synthesis than saline control (1.58±0.14 vs. 0.34±0.05 nmol/lO 9 platelets, p<O.O01), whereas native HSA did not (0.44±0.06). In the thromboxane B 2 production, glycosylated HSA caused a much greater increase (1.26±0.23, p<O.O05) than native HSA, while glycosylated LDL demonstrated no significant change (1.80±0.07) compared with native LDL. On the other hand, glycosy- lated HSA strongly suppressed arachidonic acid-induced prostacyclin production from endo- thelial cells compared with native HSA (0.91±0.03 vs. 2.56±0.27 ng 6-keto PGFlo/ml medium, n=6, p<O.O01). These data would suggest that glycosylation of serum proteins play a role in arachidonic acid metabolism in platelets and endothelial cells.

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POS-003-093 GLYCOSYLATED CARBONIC ANHYDRASE I IN ERYTHROCYTES FROM PATIENTS WITH DIABETES MELLITUS

K MURAKAMI, Y OHTSUKA, M SHIMADA, T KONDO, Y. KAWAKAMI First Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan

A glycosylated form of carbonic anhydrase isozyme I (CA-I) was found in human erythrocytes of diabetics. Both of glycosylated and nonglycosylated forms of CA-I were purified from human erythrocytes. The glycosylated CA-I was isolated using boronate affinity chromatography. Levels of the glycosylated CA-I purified from erythrocytes taken from diabetics were significantly higher than those from normal controls. The ratio of glycosylated CA-I to total CA-I was strongly and positively correlated with HbA1c levels. The specific activity and the immunological activity of the glycosylated CA-I were lower than those of the normal enzyme. The susceptivity of the glycosylated CA-I to ~-chymotrypsin decreased more than that of normal CA-I. Incubation of the nonglycosylated CA-I with [~H]-D-glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein. It is suggested that exposure of red cells to higher concentration of glucose in diabetes bringsabout glycosylation of red cell enzyme proteins.

POS-003-094 DEVELOPMENT OF A HbAIc-SPECIFIC MONOCLONAL ANTIBODY AND AN ENZYME- IMMUNOASSAY FOR HbAlc

J. ZEUTHEN, L. ANGELO, M. LAURITZEN# and V. KRUSE Novo Research Institute and Immunotechnology, Novo Industri A/S, DK-2880 Bagsvaerd; #Hvid~re Hospital, DK-2930 Klampenborg, Denmark

Current chromatographic and electrophoretic methods for measurement of HbAlc all have a number of practical or theoretical drawbacks. The objective of the present study was to develop a monoclonal anti- body (MAb) with specificity for HbAlc and to employ it in an enzyme immunoassay (EIA) for HbAlc. Two MAbs reacted preferentially with HbAlc. One of these (HEM 13FI, an IgGl) appeared to be specific for HbAlc.The specificity of HEM 13FI was defined by binding of the synthetic glycated N-terminal heptapeptide and the tryptic N-terminal octapeptide fragments of the HbAlc beta-chain. An EIA is based on the attachment of erythrocyte lysate to wells of immunoplates. Biotin-labelled HEM 13FI detects HbAlc, and bound antibody is detected by avidin-peroxidase and o-phenylene diamine as the substrate. Absorbance (492 nm) is used for the calculation of % HbAlc on the basis of calibrators with known HbAlc content. Total precision of the assay is 4-7% CV. This procedure was com- pared to isoelectric focusing (r=0.93), affinity chromatography (r=0.93) and ion-exchange chromatography (r=0.84). The present HbAlc EIA based on the HEM 13FI monoclonal antibody is a novel immunoassay specific for the glycated N-terminus of the beta-chain of HbAlc. The precision is satisfactory and the method allows processing of large batches of samples in a single assay run.

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POS-003-095 EVALUATION OF SERO~ FROLYO8~I~ AND [{bAlc IN %'REENING FOR DIABETES ~LLI~S

- THE USEFLq.,LNESS OF LINEAR DISCRIMINANT F[INL-TION

K. TANIGUCHI, K. KIJ'NIHIRO*AND Y. ORIBE~

OITA ADULT SECURITY INSTIIXJTES * FACULTY OF FIRST DEPAR'BIENT OF ~DICINE, MEDICAL COLLEGE OF OITA*~

The present study was designed to compare the usefullness of serum fructosa~ine(FA) and Hbhlc de-

terminations in screening for diabetes mellitus. All subjects, 100 men and 12 women, were classified

into three groups by 75g0GTr. Normal group(N) was consisted of 56 persons, borderline group(B)of 51

persons and diabetic group of 8 persons. Mean fasting plasma glucose value was 96.8+-6.2a~,/dl in N,

104. 0+-8. 7~/dl in B and 159. 9+-42. 2n~dl in D. Wean serum fruetos,mine value was 2.74+-0.21rmol/l

in N, 2.87+-0.20mmol/1 in B and 3.74+-0.57maol/I in D. Wean HbAlc value was 5. 06+-0. 29~ in N, 5.36+

[).45~; in B and 7. 21+1.58r, in D. Then, all subjects were classified into two groups. No diabetics

was consisted of N, diabetics was consisted of B plus D. In these two groups, two equations were

acquired as fol lows, ZI=0. 203×FPG+5. 062×FA-42. 523, 7_.2=0. 202:/FI"G+2. 434><h'bAlc-40. 830. If Z1 (Z2) is

smaller than -6.8(-8. 1), these equations are available in both specificity and sensitivity in screen-

ing for diabetics.

POS-003-096 ALBUMINURIA AND COLLAGEN-RELATED FLUORESCENCE IN EXPERIMENTAL DIABETIC NEPHROPATHY: EFFECTS OF AMINOGUAN/DINE

G J LAYTON, L McKINLEY, M COOPER, G SERUMS Endocrine Unit, Department of Medicine, Austin Hospital, Heidelberg, Australia, 3084.

It has been suggested that collagen-related fluorescence (CF, excitation at 370 nm and emission at 440nm) reflects advanced tissue glycation and that this is inhibited by aminoguanidine (A) (1). However, the contribution of advanced tissue glycation to nephropathy is not known. This study has examined the effect of A on albuminuria and renal CF in the streptozotocin diabetic spontaneously hypertensive rat, which is known to develop albuminuria after 12 weeks and renal structural changes after 32 weeks (2). Rats were randomised into 4 groups: 2 groups of control rats, untreated (C) or treated with A, 10mg/day in drinking water (CA), and 2 groups of diabetic rats, untreated (D) or treated with A, (DA). Albuminuria measured by radioimmunoassay at 0 and 32 weeks is shown as median and range (mg/24 hrs), * p<0.01, vs. nondiabetic, 1 p<0.01 vs. week 0:

C (n=7) CA (n=6) D (n=6) DA (n=6) week 0 0.09 0.13 0.11 0.17

(0.05 - 0.19) (0.10 - 0.16) (0.04 - 0.41) (0.09 - 1.0) week 32 4.71" 3.91 25.8"1. 16.2"1.

(0.82 - 13.6) (0.72 - 19.9) (18.1 - 104.8) (1.5 - 92.1) At week 32, rats were sacrificed and one kidney was homogenised in phosphate buffered saline. After removal of

salt soluble and acetic acid soluble material, collagen was extracted with type VII collagenase (lmg/ml) and proteinase K (lmg/ml) for 18 hls at 37°C (3). In 13 control and 8 diabetic rats this process solubilised 2.3i-0.2 and 2.0-&-0.3 IXmol hydroxyproline/gm kidney respectively. CF/~mol hydroxyproline was significantly increased in diabetic rats compared with control rats (43.4.+.5.0, n=8 vs 30.3+3.3, n=13, p=0.03). In individual rats at 32 weeks there was no significant relationship between CF and albuminuria in control (r = 0.13) or diabetic rats (r = 0.16). Neither albuminuria nor CF were influenced by A in control or diabetic rats. Although diabetes is associated with an increase in renal CF, the roles of advanced tissue glycation and A as an inhibitor of the process in diabetic nephropathy are unclear.

(1) Brownlee Met al, Science, 232, 1629 (1986). (2) Cooper ME et al. Am J HT (in press, 1988). (3) Monnier VM et al, Proc Natl Acad Sci, 81,583 (1984).

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POS-003-097 PROTEIN CROSS-LINKING OF CIRCULATING SERUM ALBUMIN, BUT NOT ENDOTHELIAL STRUCTURAL PROTEINS, INHIBITS ALBUMIN-MEDIATED ESTRADIOL TRANSPORT ACROSS THE RAT BLOOD-BRAIN BARRIER IN VIVO.

W. CEFALU, M. BROWNLEE, K. PRATHER Department of Medicine, Tulane University, New Orleans, Louisiana, USA and Albert Einstein College of Medicine, Bronx, New York, USA

Albumin-bound estradiol (E2) is freely available for transport across the rat blood-brain barrier (BBB) owing to enhanced dissociation of Ez from albumin without significant exodus of protein from plasma. This dissociation may involve a conformational change of albumin at the endothelial interface releasing hormone across the capillary wall. Factors such as non-enzymatic attachment of glucose (glycosylation) to human serum albumin (HSA) alter the conformation and result in inhibition of albumin-mediated transport. This change in E2 transport secondary to hyperglycemia may be due to protein cross-linking as measured by advanced glycosylation products (AGP). The present studies assess whether aminoguanidine, by inhibiting AGP formation of albumin and endothelial structural proteins, can alter albumin-mediated E2transport in vivo. To assess if AGP formation of serum albumin plays a role in transport, HSA solutions were incubated with 30 mM glucose (Glyc-AIb) and 30 mM glucose and aminoguanidine (AGP/Inhib-AIb) and 30 mM aminoguanidine.only (Control). Bioavailable E2 was determined in control rats by comparing the extraction of 3H-Es after carotid artery injection and found to be 48+4% for control, 47+_2% for AGP-Inhib/AIb (p=ns) and 39-~_2% for Glyc-AIb (p<.05). AGP (expressed as relative fluorescence/rng albumin) were found to be 1.4+_.1 for control HSA, 2.7+.2 for AGP-Inhib-AIb and 7.7+.1 for Glyc-AIb. To assess if AGP formation of endothelial structural proteins alters albumin-mediated transport, we determined E2 bioabvailability in the presence of control HSA in 4 treatment groups. After diabetic induction with streptozotocin (50mg/Kg I.V.) and verification of diabetic state (Glucose > 200 mg%), the treatment groups were as follows: Diabetic and age-matched control rats treated with aminoguanidine (25mg/Kg-day ip X 16 weeks) and diabetic and age matched control rats without treatment (X 16 weeks). There was no difference in brain bloavallabllify of albumin-bound E2 within the 4 groups. Cone. Glycosylated albumin incubated with aminoguanidine (AGP/Inhib-AIb) shows no difference in uptake of E2 when compared with control in contrast to glcosylated albumin (Glyc-AIb). As there was a 2.5-fold difference in the amount of AGP between AGP/inhib glycosylated albumin and glycosylated albumin (p<0.001), this suggests that advanced glycosylation of human serum albumin resulting in protein cross-linking may play a role in hyperglycemia-induced inhibition of albumin-mediated E2 transport. Advanced glycosylation of endothelial structural proteins on the BBB does not interfere with albumin-mediated E2 uptake.

POS-003-098 EVALUATION OF FRUCTOSAMINE IN DIABETICS.

S PLOYBUTR, A VICHAYANRAT, W NITIYANANT, S VANNASAENG, T PIRAPHATDIST, S HARN~ONG Department of Medicine, Siriraj Hospital, Bangkok, Thailand.

Measurement of glycosylated hemoglobin (GHb) has been accepted as a long-term glycemic index in DM. Recently, glycosylated plasma protein, particularly fructosamine has been shown to reflect a short- term glycemic status. Several reports demonstrated the fluctuation of fructosamine level in relation to total plasma protein concentration. %33us the usefulness of fructosamine level without correction by plasma protein is questionable. We therefore evaluate the value of fructosamine determination in various categories of diabetics andthe suitable way to express its concentration. Fructosamine was determined in 25 euglycemic subjects, 50 diabetic patients on treatment, 20 patients with diabetic ketoacidosis and 8 newly diagnosed diabetics using Fructosamine Test, Roche, Switzerland with some modification. Blood sugar (BS) was measured by glucose oxidase method. Total protein and GHb were measured by Biuret and q~BA methods respectively. The results (mean +SD) were expressed as fructosamine (FI) in mmol/l and fructosamine/protein (F2) in retool/g/10 -2 (Table). F1 showed good correlation with GHb and BS ( r = 0.8692 and 0.6535, respectively) and so did F2 ( r = 0.8506 and 0.7599). FI, F2, TP, GHb and BS of all groups of patients were significant different from normal (p <0.001). BS and fructosamine measured as F1 and F2 showed significant difference among the 3 categories of dia~tics.

Subject (n) F1 (mmol/l) F2 (retool/g/10 -2) TP (g/dl) GHb(%of total Hb) BS (mg/dl) Eugl ycemic (25) 2.69+0.13 4.01±0.29 6.72±0.44 6.36±0.74 86.6±11.3 Treated DM (50) 3.12±0.31 4.37+_0.52 7.18±0.64 9.64±2.47 166.4±57.7 DM with DKA (20) 3.70±0.47, 6.44_+1.16, 5.86_+0.98 16.50+4.21 689.7+275.4 Newly diagnosed DM(8) 4.07+_0.47 5.57_+0.84 7.35+--0.37 16.61+_3.44 319.5+_119.1 * p<0.05 newly diagnosed DM vs DKA (FI and F2)

In conclusion, fructosamine concentration whether divided by plasma protein concentration or not can be used as a valuable glycemic index in diat~tics.

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POS-003-099 ENZYME-LINKED IMMUNOASSAY" (ELISA) OF GLYCATED COLLAGEN BOUND PROTEIN IN DIABETIC PATIENTS

H KANESHIGE, H MACHIMURA, K TANAKA, S TAMURA, H SAKAI Department of Internal Medicine, School of Medicine, Tokai University, City, Kanagawa-ken, Japan

Isehara

Non-enzymatic glycation of various tissue proteins has been reported in relation to complications in diabetes mellitus. It is presently unknown if glycated collagens contribute to the deposition of immune complexes in various tissues. The aim of this study was to examine the aberration of IgG and albumin binding with collagen after in vitro glycation by the enzymelinked immunoassay (ELISA). Type 4 collagen (iO~g/ml) was added to microtiter wells and glycated with lOOmM D-glucose for iO days. After glycation, a serum sample was added to each well and IgG and albumin bound to glycated collagen was measured after reaction with HRP coupled to goat anti-human IgG and albumin and expressed as opsonic density. The IgG and albumin bound to glycated collagen after I0 days of incubation were measured in 28 diabetics and iO age-matched controls. Increasing amounts of protein bound to glycated collagen were observed in proportion to in vitro glycation of collagen. The amount of bound IgG and albumin were significantly higher in diabetics than in the controls (p<0.Ol). Significant increases of bound IgG and albumin were observed in patients with proteinuria or over iOyrs of diabetes compared with those without complication or a duration of less than iOyrs. It was suggested that the increasing amount of protein bound to glycated collagen might be related to the formation of in situ immune complex in diabetic patients.

POS-003-100 RE-EVALUATION OF THE ffRUCTOSAMINE REACTION

R. FARRANT, G. PHILLIPOU, C. SEABORN and P. PHILLIPS Endocr ine and Diabetes Serv ice, The Queen El izabeth Hospi ta l , Woodvi l le, South Aust ra l ia 5011

Or ig ina l l y repor ted by Johnson et al ( i ) the f ructosamine reaction has been advocated as a probe o f glucose homeostasis du r i ng the past 7 - 21 days. We have studied the two major methodological problems: assay s tandard izat ion and react ion spec i f ic i ty (~) Assay s tandard iza t ion . We have found that a d ispers ing agent (T r i t on X-100) is necessary to fu l ly solubi l ize the n i t ro -b lue di formazan (NBD) formed by reduct ion of n i t ro -b lue tetrazol ium ch lor ide. With the so lub i l i z ing agent :

The spectra l character is t ics of l - d e o x y - l - m o r p h o l i n o f r u c t o s e (DMF) and prote in /p lasma samples are equiva lent w i t h a max imum at 525nm. The absolute response is increased 250% and 160~ for both DMF and human serum albumin respec t i ve ly .

Reaction spec i f i c i ty . We have fu r the r noted that The react ion for DMF does not follow pseudo f i r s t - o r d e r k inet ics as noted for pro te in samples D ihydroxyace tone is a m o r e " r o b u s t " ca l ib ra t ion s tandard than DMF, especial ly with respect to pH.

We propose 1. Tha t reagents for the f ructosamine react ion using NBD as an end-po in t ind icator contain a

sui table so lub i l iz ing agent such as T r i t on X-100. 2. D ihyd roxyace tone be used as the s tandard in place o f DMF. ( i ) Johnson R . N , , Metcal f P . A . , Baker J .R. Clin Chim Acta 1982; 127:87 - 95 (= ) Johnson R . N . , Baker J .R . Clin Chem 1987; 33: 1955 - 1956. - -

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POS-O03-101 GLYCATED PROTEINS IN THE DIAGNOSIS OF GESTATIONAL DIABETES.

AAJ REICHELT, EMK RUSS0, AR CHACRA Endocrine Division, Escola Paulista de Medieina, Sao Paulo, Brazil

Because the morbidity associated with undiagnosed gestational diabetes (GDM), there is a reco~ nized need of screening programs in all pregnancy clinics. In this paper we investigate the use fulness of glycated hemoglobin (HbAI) and glycated protein (GP) in the detection of GDM. We have studied i00 normal pregnant women and 21 with GDM. The HbAI was measured by the ion ex- change method (nl 6,3 to 8,6%) and GP by the affinity chromatography (nl 0,8 to 2,4%). The results of fasting blood sugar (FBS), HbAI and GP were compared with the OGTT.

SENSIBILITY SPECIFICITY ACCURACY PREDICTIVE VALUES

POSITIVE NEGATIVE

FPG >105 mg% 78% 99% 96% 93% 96%

HbAI > 8,6% 67% 88% 85% 50% 94%

> 9,4 33% 94% 86% 50% 89%

GP > 2,4% 27% 88% 79% 29% 87%

> 2,7% 16% 98% 86% 60% 87%

In conclusion, the measurement of glycated proteins (HbAI and GP) is not useful in the diagnosis of gestational diabetes mellitus.

POS-003-102 CLINICAL USEFULNESS OF COMBINATION ASSAY OF FRUCTOSAMINE AND FPG AS A SCREENING TEST FOR DIABETES MELLITUS

S. YAMASAKI, S. EGASHIRA Itoshima Medical Association Hospital,Atherosclerosis Res.& Metabolic Res. Fukuoka, Japan

Recentry, Fructosamine(FRA) has been noticed as a clinical indicator of Diabetes Mellitus(DM). We have studied the cut-off line by the combina- tion assay of FRA & FPG for the screening of DM.

The upper limited line of FRA is estimated at 2.81 mmol/l from the result of determination of normal subjects, 2.63 ± 0.18 mmol/l; 120 mg/dl reco- mmended by WHO is used as the upper limited line for FPG. The sphere surrounded by the limited value of FPG(X-line) and also FRA(Y-Iine) is estimated to be the normal range. Patients having abnormal values of either or both indicators are classified into the borderline group. All of the normal subjects are distributed in normal range and also 100% of DM cases are distributed in the abnormal range. On the other hand, the borderline group is divided into two groups; namely 23.1% of normal cases are distributed in the normal range and 76.9% are in the abnormal range.

In conclusion, OGTT can be omitted for 71% of the cases when we employ the above mentioned combination assay for screening of DM. Besides, the measurement of FRA can easily be performed by autoanalysis for handling a number of samples. Therefore, FRA is available as an indicator for mass health check for DM. These facts will highly contribute to find the latent diabetic patients in the field.

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POS-003-103 AN ASSESSMENT OF SERUM FRUNCTOSAMINE AND BLOOD GLYCATED HEMOGLOBIN MEASUREMENTS FOR THE SCREENING OF GLUCOSE INTOLERANCE

JUI-SAN CHEN DEPT. OF CLINICAL PATHOLOGY, NATIONAL TAIWAN UNIVERSITY HOSPITAL, TAIPEI, TAIWAN,

D.M. 32 (6.]9) ]GT 37 (7.]6) Normal 349 (67.5) Unclass 99 (]9.2) Total

Serum fructosamine, blood hemoglobin AIC and A I, fasting and 2 hours postprandial plasma glucose, 75 gm oral glucose tolerance test (OGTT) were performed on adult non-pregnant physical examinees for the screening of glucose intolerance. All tests were accomplished on the same day of the blood sampling. According to the official guide to diagnosis and classification of diabetes mellitus and other categories of glucose intolerance prepared by the National Diabetes Data Group, NIH, and American Diabetes Association Statistics Committee, January 1980, the examinees were classified and the results were :

No.ease (%) F~uctosamine m mol/l HbAIC (%) HbA] (%) mean Range <2.9ease(%) mean Range <6. ]%case(%) mean Range <9.]%case (%) 3.60 2.5-6.28 30 (93.8%) 8.49 4.7-16.0 29 (90.6) 12.6 7.1-20.6 29 (90.6) 2.7] ].96-3.6 8 (21.6%) 5.30 4.]-7.8 5 (13.5) 10.2 6.5-]].4 6 (16.2) 2.52 ].2-3.1 6 (].72%) 4.52 2.7-6.0 0 (0) 7.l 4.7-]0.6 5 (].43) 2.52 1.5-3.1 7 (7.07%) 4.69 3.5-6.7 2 (2.0) 7.3 5.5-9.4 3 (3.03)

5]7 eases (100)

DM: Diabetes mellitus ]GT: Impaired Glucose Tolerance Unclass: Unclassifide

POS-003-104 USEFULNESS OF MEASUREMENT OF PLASMA FRUCTOSAMINE LEVELS IN DIABETICS.

H.BESSHO, Y.NOMURA, Y.KADOYA, H.SASAKI, T.OHBOSHI, M.KONDO*, T.SANKE*, K.NANJO*, K.MIYAMURA* Department of Meicine, Ozaki Public Hospital, *The First Departmwnt of Medicine, Wakayama University of Medical Science, Wakayama Japan The usefulness of measurement of plasma fructosamine (FR) levels in diabetics was evaluated. (I) Influence of three anticoagulants (EDTA-2K, heparin, and NaF) on FR: Plasma FR levels varied depending on the kind of anticoagulants used. Compared with EDTA-2K and heparin, the lowest FR level was noted in the presence of NaF. While all plasma FR levels were low compared with serum FR levels, a significant positive correlation was observed between both (p<O.O01). (2) Clinical usefulness of plasma FR levels using EDTA-2K as an index of blood glucose control: Plasma FR levels in diabetics (n=204) were 3.51±0.05 mmol/l (mean±SE), which was significantly higher than 2.5?±0.02 mmol/l in healthy subjects (n=57) (p<O.01). Plasma FR levels showed a significant positive correlation with simultaneously measured levels of fasting plasma glucose (FPG), HbA1c, and glycated albumin by affinity chromatography, and also with average FPG levels in past two weeks (p<O.O01). According to FPG levels, diabetics were divided into good control group with less than 125 mg/dl, fair control group with 125-150 mg/dl, and poor control group with more than 150 mg/dl. In these three groups, plasma FR levels were 2.90±0.08, 3.19±0.07 and 3.73±O.07mmol/1, respectively; these values in three groups were significantly differnt each other (p<O.01). (3) Comparison between plasma FR and HbA1c levels under influence of short-term variations in plasma glucose levels: ?5g-OGTT was performed in healthy subjects (n=16) and diabetics (n=54) to determine plasma glucose (PG), HbAlc, and plasma FR levels, time- dependently. Based on the increment of PG, a PG(60'), from the basal value to 60 min-values after loading, the subjects were divided into three groups: A (100-149 mg/dl), B (150-199 mg/dl), and C (200-250 mg/dl). C group showed a significantly higher values of ~ HbA1c(60') than A and B groups (p<O.01), whereas there was no significant difference in ~FR(60') values among three groups. These findings demonstrated that the determination of plasma FR levels is useful as an index of blood glucose control and is also exempted from any restraint of blood sampling time, being free from the influence of short-term variation in blood glucose level.

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POS-003-105 REAGENT FOR THE IMPROVED COLORIMETRIC SERUM FRUCTOSAMINE ASSAY

BW VOGT, L KERSCHER, J SIEDEL, J ZIEGENHORN Boehringer Mannheim GmbH, Biochemical Research Center D-8132 Tutzing, Federal Republic of Germany

The assay of glycated serum protein (fructosamine) as an index of diabetes control may be easily conducted with a reagent described by Johnson et al. (I) based on the reductive color formation from nitroblue tetrazolium (NBT) by ketoamines in alkaline medium. This method, however, suffers from various inter- ferences, mainly due to lipemia, uric acid and protein matrix effects.

We modified this reagent by adding a detergent mixture (22g/L), uricase (4U/mL) and raising the NBT concentration to 0.6mmol/L. The detergent system rapidly clarifies even strongly lipemic samples, and, in addition, improves the solubility of the formazan dye and eliminates quenching of the color signal from desoxymorpholinofructose (DMF) when added at constant amounts to solutions with increasing albumin concentration (20-100g/L), suggesting removal of protein matrix interferences. Uricase effectively prevents uric acid interference.

With sera from healthy and diabetic individuals, a comparison of this reagent and of that according to (I), applied to a Hitachi 704 analyzer (20NL sample/ 350~L reagent, kinetic measurement from 9 to i0 min at 546nm/37°C) against a NPLC-method determining furosine after acid sample protein hydrolysis (2) gave an improved correlation coefficient (r=0.98 vs. 0.91 with reagent (I)).

We conclude that the modified reagent might distinctly contribute to an improved performance of routine serum fructosamine assays.

i. Johnson RN et al., Clin. Chim. Acta 1982;127:87-95

2. Schleicher E et al., J.Clin. Chem. Clin. Biochem. 1981;19:81-87

POS-003-106 LENS FLUORESCENCE VERSUS HEMOGLOBIN GLYCOSYLATION

AND LONG-TERM DIABETES COMPLICATIONS

M LARSEN, B KJER, I BENDTSON, P DALGAARD & H LUND-ANDERSEN Gentofte Hospital and Steno Memorial Hospital, Copenhagen, Denmark

A non-invasive study of lens fluorescence in selected young insulin- -dependent diabetics (mean age 30 years, mean duration 5 years, n = 36) revealed that well-controlled diabetics (mean HbAlc since onset lower than 7.0 %) had less fluorescent lenses than poorly controlled diabetics (mean HbAlc since onset higher than 9.7 %; P = 0.001). A higher lens transmission was also found in patients with low HbAlc. Thus, the aging of the lens, which is markedly accelerated in diabetes, appears to be delayed by good metabolic control.

In patients with a longer duration of diabetes (mean age 46 years, mean duration 30 years, n = 22), lens fluorescence did not vary significantly with the presence or absence of long-term complications (nephropathy, hypertension, retinopathy). The lens transmission was, however, signi- ficantly lower in patients with complications. A correction of the fluorescence that takes this decrease in lens transmission into account reveals that patients with complications had a slightly higher lens fluorescence than those without complications.

The interpretation and implications of the cited findings are discussed.

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POS-003-107 CLINICAL USEFULNESS OF SERUM FRUCTOSAMINE LEVELS IN DIABETICS

H. KURAHACHI, K. MORIDERA, T. ISHIHARA AND T. IGARASHI DEPARTMENT OF INTERNAL MEDICINE, KOBE CITY GENERAL HOSPITAL, KOBE, JAPAN

Serum fructosamine (FRS) levels in diabetics were measured by the Roche ~ "Fructosamine" test, in which coefficient variations of intraasay and inter- assay were 1.0 and 1.7%. The average basal FRS levels were 2.56±0.23 (m±SD) in the healthy controls (n=395), 3.37±0.42 in one group of diabetics (HbAI~I0% , n=120) and 4.42±0.64 mmol/L in another group of diabetics (HbAI>I0% , n=50). The differences between the three groups were highly significant (p<0.001). FRS correlated with fasting blood sugar (r=0.643) and HbA 1 (r=0.825). Effect of difference of age or sex on the basal FRS levels was hot found.

Clinical observations in the diabetics including diabetic coma and unstable diabetes revealed that FRS reflects the average blood sugar levels of approxi- mately two weeks preceding its determination. Slight diurnal, postprandial variation of FRS levels were observed in some cases, although rather in- constantly.

FRS was significantly elevated in the presence of high serum creatinine (>2.5 mg/dl) or birilubin levels (>3.0 mg/dl), while it was significantly low in the presence of hypoalbuminemia (<3.0 g/dl). The explanation for these observa- tion need to await further study, but in the absence of these abnormalities in blood chemistry it was suggested that FRS determination may be more useful in monitoring short-term, i.e., approximately two weeks blood glucose control than HbAI, and that the combination of FRS and HbAI can provide useful information regarding the efficacy of therapy, particularly in patients with unstable diabetes.

POS-003-108 GLYCOSYLATED LOW DENSITY AND HIGH DENSITY LIPOPROTEIN CONCENTRATION IN PATIENTS WITH NON- INSULIN-DEPENDENT DIABETES MELLITUS.

MT FUH, DA WU, CY JENG, DC SHEN, GM REAVEN, AND Y DI CHEN Department of Medicine, Tri-Service General Hospital, Taipei, Taiwan, & Stanford University, Stanford, CA, USA

Although there is good evidence that patients with non-insulin-dependent diabetes mellitus (NIDDM) have abnormalities of plasma l ipoprotein concentration, there is re la t ive ly l i t t l e in- formation concerning possible changes in l ipoprotien composition in these individuals. In parti- cular, the poss ib i l i ty that l ipoproteins may become glycosylated to an abnormal degree in NIDDM has not been evaluated. Therefore, we measured glycosylated LDL and HDL (nmoles glucose/mg pro- tein) in sex, weight and age-matched patients with NIDDM (n=27) and control subjects (n=32). Fasting plasma glucose levels and glycosylated hemoglobin (% HbAI) were also determined in these patients. Patients with NIDDM had higher mean (~ SEM) concentrations of fasting plasma glucose (177+19 vs 90+7 mg/dl, p<O.O01), HbA 1 (10.2+0.3 vs 7.8+0.12%, p<O.O01), glycosylated LDL (65.4+39-vs 35.1+7.0 nmoles glucose/mg LDL proteTn, p<O.OOT) and glycosylated HDL (4.9+ 2.3 vs 2.4+I.4 nmole~glucose/mg HDL protein, p<O.O01). Signif icant correlations were noted between magnitude of fasting hyperglycemia and degree of both glycosylated DLD (r=0.57, p< 0.001) and glycosylated HDL (r=0.56, p<O.O01). Similar correlation were also seen between con- centration of either glycosylated LDL (r:0.48, p<O.O01) or glycosylated HDL (r=0.46, p<O.O01) and glycosylated Hb. Of interest was the observation that LDL was glycosylated to a greater degree than HDL by approximately 15-fold, and this was ture of both patients with NIDDM and normal subjects. Thus, apoB appears to be more susceptible to glycosylation than is apoAl+ Al l . In conclusion, paitents with NIDDM have elevated concentrations of glycosylated LDL and HDL, are the degree of the elevation is d i rect ly correlated with severity of hyperglycemia. To what extent these changes in l ipoprotein composition contribute to the abnormal catabolic behavior of LDL and HDL that has been described in patients with NIDDM remains to be defined.

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POS-003-109 CLINICAL EVALUAIION OF IHE ASSAY OF GLYCOSILAIEO SERUM ALBUMIN (GA)

R. WAGNER, G. BENKCR, H.J. KRONKE, D. REINWEIN University of Essen (Department of Clinical Endocrinology), Germany (FOR)

In Diabetes mel]itus, the concentration of GA is known to correlate with mean blood sugar levels similar to HbA1, although i t ref lects shorter periods of metabolic control. We have studied GA and HbA1 in d i f ferent c l in ica l situations (improvement of metabolic control following structured patient [nstruct ionl renal fa i lure; para- proteinemia) in order to elucidate the advantages and disadvantages of both methods. 1. In 30 patients with insulin~dependent diabetes mell itus (IDDM), we have performed HbA1 (chromatographic method, Boehringer) and GA (Fructosamine~ Roche) determinations before and 6 month after part icipation in a structured diabetic teaching programm (SOTP). 2. To investigate the influence of renal fa i lure, we have performed HbA1 and GA estimations in 35 patients with terminal renal fa i lure without IDDM, in 11 patients with IODN and renal fa i lure and in 23 healthy volunteers. 3. The influence of serum protein concentration on GA was investigated in Zl patients with paraproteinemia (PP) without IDDM. The results are summarised in the table below:

SOIP NEPHROPATHY PP before after p IDDM not IDDM volunteers

HbA1 (%) 11.4+ 0.6 9.Z+1.5 0.001 11.9+1.7 9.4,1.4" 7.} ,0.6 - GA(mmol/l) 3.6~ 0.5 2.9~0.? 0.02 3.2;0.~* 1.1~0.4 1.3;0.5 4.3+I" * p 0.01 compared to healthy volunteers Conclusion: 1. Similar to UbA1, the measurement of GA can be used to monitoringimproving metabolic control in IODM. 2. In terminal renal failure, HbA1 (chromatographic methods) is carbamylated leading to elevated levels whereas GA remains reliable in this situation. 3. In patients with paraproteinemia without IDDM, GA is elevated presumably due to gtycosyiation of paraproteins.

POS-O03-110 CHANGES IN LEVELS OF GLYCATED PLASMA PROTEINS IN NORMAL PREGNANCY

A.E. OHWOVORIOLE, J.O. OLORONDU, E.E. EMUVEYA~, AND A.O. ABUDU Departments of Medicine and Obstetrics and Gynaecology, College University of Lagos, Lagos. Nigeria

of Medicine,

Carbohydrate (CHO) tolerance in pregnancy has been variously reported to be impaired or enhanced using glucose tolerance tests. Use of glycated proteins such as glycated haemoglobin (GHb) or glycated plasma proteins (GPP) should be informative in assessing the pattern of CHO metabolism at the various stages of pregnancy. GPP have a shorter half-life than GHb and are a better index of CHO metabolism in the intermediate term. In this study we assessed CHO metabolism in pregnancy by quantifying the levels of GPP at various gestational ages. GPP levels were estimated by a modification of the tbiobarbituric acid colorlmetric method. In early pregnancy, there was a fall in GPP levels compared to non pregnant controls. The mean levels of GPP then rose to a peak in late third trimester. Total plasma protein {TPP} levels fell steadily throughout pregnancy and failure to correct for the falling levels of TPP gives falsely low GPP levels and thus a false impression that GPP levels fall in pregnancy. Correlation between GPP and fasting plasma glucose levels was weak in early pregnancy but became moderately high and significant in late pregnancy. The results of this study show that there is enhanced CHO metabolism in early preganancy and confirms the generally accepted view that there is increased CHO intolerance in late pregnancy

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POS-O03-111 TUBULIN GLYCOSYLATION MAY CONTRIBUTE TO DELAYED REPLICATION OF CULTURED HUMAN ENDOTHELIAL CELLS

GROWN IN HIGH GLUCOSE CONCENTRATIONS.

M LA SELVA, *M PORTA, *P MOLINATTI, GM MOLINATTI.

Cattedre d~ Clinics Medica B e *Endocrinologia, University of Turin, Turin, Italy.

Data from this and other laboratories suggest that the replication of cultured human umbilical

vein endothelial cells (HUVEC) is delayed in the presence of supra-physiological concentrations

of glucose. Delay appears to occur at the G2 phase of the cell cycle, i.e. between DNA synthesis

and mitosis. Since tubulin de-polymerization and polymerization provide the basis for the

formation of the mitotic spindle, we aimed at investigating whether non-enzymatic glycosylation

may lead to abnormal function of this protein and thereby to impaired cell division.

HUVEC were cultured to confluency in supplemented M-199 medium, subdivided in multi-well

plates and grown in secondary cultures in the presence of glucose I00 or 900 g/l. Six days

later colchicine O, 5, I0, 20 and 40 mg/l was added for 30 min and afterwards the HUVEC

were fixed by cold methanol and treated with dithio-bis-succinimidyl propionate to stabilize

microtubules and Triton X-IO0 for membrane permeabilization. The cells were then stained

for indirect immunofluorescence, using a mouse anti-tubulin antibody and an anti-mouse

fluorescein-conjugated rabbit antiserum.

Preliminary results show that the microtubular network as well as cell shape and integrity

were subverted to a lesser degree by colchicine 5 to 20 mg/l in HUVEC grown in glucose 900

g/l than in those grown in i00 g/l.

This observation suggests that supra-physiological concentrations of glucose may render tubulin

less prone to colchicine-indueed de-polymerization. Similarly to what reported for peripheral

nerve fibres, non-enzymatic glycosylation might render microtubules more stable and therefore

delay the formation of the mitotic spindle.

POS-003-112 SUPPRESSIVE EFFECT OF ~-KETOALDEHYDE DEHYDROGENASE ON THE ADVANCED PROCESS OF THE MAILLARD REACTION.

F HATA, N IGAKI, T NAKAMICHI, S MASUDA, S NISHIMOTO, M OIMOMI, S BABA, H KATO* The 2nd Dept. of Internal Medicine, Kobe University School of Medicine, Kobe, Japan Dept. of Agricultural Chemistry, The University of Tokyo, Tokyo, Japan*

Advanced-stage products of the Maillard reaction are accumulated in the tissue and form cross- links. Their relationship between aging and the development of diabetic complications has drawn attention. Especially, 3-deoxyglucosone (3-DG), as an intermediate active substance of the Maillard reaction increases the fluorescence of the advanced Maillard products. Therefore, we have investigated the effect of e-ketoaldehyde dehydrogenase (e-KAD), which oxidizes 3-DG, on the Maillard reaction.

Alfa-KAD was extracted from human liver obtained at autopsy. Enzyme activity and Km value were determined by absorptiometry via the reduetive reaction of NAD using methylglyoxal or S-DG as substrates. The character-istics of the enzyme were examined using SDS-PAGE. The 3-DG was allowed to react with a-KAD and the resultant product was incubated with lysozyme at 37aC for 2 weeks. The intensity of fluorescence of the advanced products of the Maillard reaction was determined at an excitation wavelength of 370 nm and an emission wavelength of 440 nm before and

after the incubation. The Km value of the enzyme was determined using methylglyoxal as a substrate was 0.24 mM.

The molecular weight was about 43000. When 3-DG pretreated with e-KAD was incubated with lysozyme, the intensity of fluorescence was significantly suppressed as compared with that of 3- DG untreated with a-KAD.

It was suggested that the action of a-KAD is involved in the regulation mechanism of the Maillard reaction in the body and plays an important role in the development of diabetic com- plications.

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POS-003-113 A NEW METHOD FOR PLASMA GLYCATED ALBUMIN WITH HPLC

K SHIMA,M HIROTA,F ABE Department of Laboratory Medicine, Tokushima University Medical Scool, Tokushima, Japan

As one of the indicators for long term diabetic control, glycated hemoglobin(HbAlc) has been widely employed to imply the plasma glucose level for a duration of approximately one to two months. However, an indicator able to reflect the diabetic state for a shorter duration than HbArc is in need, and plasma glycated albumin(GA) could be one of the candidates for this purpose. However, the various methods for measuring this substance reported so far are not simple nor precise. We have develop an assay for serum glycated albumin with HPLC. Method: In this assay system, albumin is fractionated by an ion exchange chromatography and then the fractions of albumin are automatically applied to a boronate column and separated into GA and non GA fractions. The GA value is expressed as the percentage of total albumin. I O~1 of serum is needed for an assay, of which procedure completes within 20min. Results: The concentrations of glycated protein in GA and non GA components thus fractionated were 12.5 and O.14nmol/mg protein when measured by RIA using a monoclonal antibody to glucitol lysine. When HSA was incubated with 27.8mM glucose, the percentage of GA was increased from 22~ before to 39 and 50~ after the incubation for 14 and 47 days, respectively. Both the intra- and inter-assay precision were satisfactory: CV; 1.5-3.5~ and 2.8-7.5~, respectively. The GA values obtained from 50 normals, 40 IDDM and 25 NIDDM were 22.0 ± 1.8, 39.6 t 5.A and 39.4 t 5.9(M i SD)Z, respectively. The values of the diabetic groups were significantly higher than that of the normal(p < 0.001). The GA values obtained from NIDDM were well correlated

with those of HbA, c(r = 0.76) but not so well with those of FBS(r = 0.39). Conclusion: This newly deviced method for serum glycated albumin is simple, specific and

precise enough to be applied to the clinical use.

POS-003-114 RELATIONSHIP BETWEEN ADVANCED-STAGE PRODUCTS OF THE MAILLARD REACTION AND THE DEVELOPMENT OF DIABETIC COMPLICATIONS.

M OIMOMI, Y MAEDA, T NAKAMICHI, S MASUDA, F HATA, S NISBIMOTO, H HATANAKA, S BABA The second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan.

The advanced-stage products of the Maillard reaction are accumulated and form cross-links in long-lived proteins in the body tissues. Mallard reaction, therefore, has drawn attention in relation to the etiology of diabetic complications. We selected lens protein as a long-lived protein and investigated the relationship between advanced-stage products of the Maillard reaction and diabetic complications. The cataractous lens was separated into the capsule, cortex and nucleus. Each part was

hydrolyzed with 6 N MCl and used as a sample. The advanced product (peak L I) of the Maillard reaction analyzed by fluorescence HPLC (excitation wavelength 370 nm, emission wavelength 440 nm). The grade of diabetic retinopathy was determined after the resection of the cataract. The peripheral nerve conduction velocity was used as an index of diabetic neuropathy. In both diabetics and non-diabetics, the peak L 1 level in the cataractous lens was the highest

in the nucleus, followed by the cortex and capsule in this order. The peak L 1 level in the lens nucleus was significantly higher in diabetics with complications than in diabetics without complications. Comparison of the age-and duration-matched background and proliferative retinopathy groups showed that the peak L1 level was significantly higher with progression of retinopathy. Peak L 1 tended to increase in proportion to the decrease of peripheral nerve conduction velocity in diabetics. It was indicated that taking into consideration that the advanced-stage products of the

Maillard reaction have cross-links, those in human tissue are important in the development and progression of diabetic complications.

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