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1948 CL IN ICALCHEM ISTRY,Vo l. 27 , N o. 11, 1981

Fig. 1 . AutoAnalyzer flow diagram in-c lu din g flo w r ate sand rap id (6 .30 mm ) method for usew ith large n um be r o f s amp le s (40/h) bymeans o f a T ec hn ic on A uto An aly ze r II .F ig ure 1 s ho ws th e flow c ir cu it d ia g ram .The coloration o f d ia ly ze d 5-hydroxy-methylfurfural (5-HMF) is developed inan heat ing bath (7 5 # {1 76 }C )n 2.20 mm . Thecomposi t ion of color re ag en t (ju stifiedin Figure 2) is : th iobarb itu ric ac id(TBA) 0.02 mmol/L , oxa lic acid 1mmo l/L , B rij 35 5 mL/L . The solu-tion , stored in a brown flask , is stable fo rat least one month at room tempera-ture.

For GHb assays, the dialyzed hem o-lysates w ere ad justed to 10 g/L, an dhydrolyzed by the technique of Pecoraroet al. (4): 1 m L of hem olysate w as incu-bated in a heating block for 5 hat 98#{176}Cw ith 1 mL of oxalic acid solution (1mmol/L , containing 2 mL of B rij 35per liter), in a screw-cap Te flon via l.Fo r GSP a ss ay s s erum was dialyzedfo r 18 h at 4 # {1 76 }Cs pH 7.4 buffer [N aC 10 .1 5 m mo l/ L, 4-(2-hydroxyethyl)- 1-piperazinethanesulfonic acid (HEPES)0.05 m mol/L1, then w as hydrolyzed bythe McFarland et al . technique (2): 0. 2mL of serum was dilu ted to 2 mL w ithd istilled w ate r and m ixed with 1 mL of

F ig . 2 . P ara mete rs o f th e 5-H MF colo ra -tion developm ent: (a) tem perature ofheating bath , (b ) TBA concen tra ti on , (C )o xalic a cid c on ce ntratio nR esults expressed as peak height In arbitraryunits

F ig . 3 . A u toAna ly ze r tra cin g fo r 5 -HMFstandard c urv e n orm al b lo od (befo re d i-a ly sis : g lu co se , 3.7 mmol/L; HbA1,5 .8% ; se ru m pro te in s, 67 g /L ), a nd dia-be ti c b lood (before d ia lys is : g lucose , 9 .3mmol/L ; HbA i, 16% ; s e rum p ro te in s , 5 9gIL)A. d ia lyzed b lank ; B. d ia ly ze d a ss ay ; C, nondlalyzedblank; D. nond la ly z ed a s sa yoxalic acid solution. E ach sam ple wasmeasured against its own NaBH4-re -duced blank (N aBH4 0 .1 3 mmol/L in -stead of distilled water fo r dilution,added 1 h before th e oxalic acid addi-tion).

5-H MF standard solut ions w ere pre-pared to cover th e r an ge 2 .3 -2 3 tmol/Lan d used th e sam e d ay.Figure 3 shows a t yp ic al strip-chartrecording. W e determined th e within-assay precis ion (n = 10,1 = 23 tmol/L,CV = 1.3%) and the between-assayprecision (GHb: n = 14,1 = 11 mol/L,CV = 4.8% ; GSP: n = 14,1 = 5.3 tm ol/L ,CV = 11%). Analytica l recovery of5 -HMF added to h yd ro ly sa te s wasabout 100% ; when added before hy-dro lysis it w as about 60% for hem oglo-bins. The discrepancy could be ex-plained by a partial decomposition of5-HM F during hydrolysis. For GHb andGSP assays the dialysis of proteins w asnecessary to rem ov e free carb oh ydrates,wh ic h in du ce d a spurious increase inapparent glycosylated proteins, asshown in F igure 3 (C , D).

We compared the manual m ethod (3 )w ith our sem i-autom ated method forGSP (n = 57, y = 1.06x - 0,45, r =0.991) and for GHb (n = 37, y = 1.04x +0.05, r = 0 .9 69 ). The values obtained bythe m odified H bA 1 chrom atographicmethod (5 ) were correlated to thoseobtained by the sem i-automated m eth-od (n = 51, y = 4,29x - 1.73, r = 0 .8 7).The mean values, expressed as nano-m oles of 5-H MF per m illigram of pro-teinwere for GHb 1.62 (SEM 0.05) and2 .2 4 ( SE M 0 .0 8) ,r es pe ct iv el y,f or 3 0n orm al co ntro ls a nd 9 0 d ia b et ic s ub je c ts(p < 0.0 01 ), a nd fo r serum p ro te in s 0 .2 1(SEM 0.02) and 0.43 (SEM 0.04),re-spectively , for the same patients (p