G$ycated Hemoglobin in Fractionated Erythrocytes

Transcription

1 CLIN. CHM. 35/6, (1989) G$ycated Hemglbin in Fractinated rythrcytes KjI Nakashlma, Osamu Nlshlzakl, Yukl Andh, Hltshl Takel, Aklra Ital, and Yukl Yshlda rythrcytes were fractinated, by centnlugatin, n the basis f cell density, which is prprtinal t cell age. Glycated hemglbin (Hb A1) in the fractinated cells was measured and cmpared with the Hb A1,, in whle bld and with the fructsamine cncentratins in plasma f nrmal and diabetic subjects. In nrmal subjects, Hb A1 increased accrding t the age f the erythrcytes, but hyperblically rather than linearly, indicating the slw cnversin f Hb A t Hb A1,, with saturability. The greatest crrelatin f Hb A1 with plasma fructsamine was in yunger middle-aged cells. A subgrup f subjects with impaired glucse tlerance had high Hb A1 in lder cells. Hb A1,, f the yung cells reflects recent glycemic status, fr mnitring the initial stage f the treatment f diabetics, whereas Hb A1 f the lder middleaged cells mre specifically reflects the lnger term glycemic status, fr screening and fr the lng-term mnitring index fr treatment f diabetes. Additinal Keyphrases: diabetes mellitus screening mnitring therapy cell age and glycatin. fructsamine Determinatin f glycated hemglbin (Hb A1,,) in whle bld is well established and widely used fr mnitring lng-term (abut tw mnths) cntrl f the cncentratin f bld glucse in knwn diabetic patients (1-8), and als as a tl fr screening fr diabetes mellitus (9-11). Jhnsn et al. (12) develped a clrimetric assay fr serum fructsamine, which riginates mainly frm the nn-enzymatic glycatin f serum prtein. This assay is becming widely used as an inexpensive, simple, shrtterm (tw weeks) index t the cncentratin f glucse in bld (13). Hwever, the assay is based n measuring the reducing activity f the glycated prtein in an alkaline ph, s ther reducing agents, which vary frm serum t serum (14), interfere. In diabetic patients, varius glycated prteins differ in their bilgical half-lives and reactivities t mtr blue tetrazlium (15), limiting the specificity f the fructsamine assay in this grup. Measurement f glycated albumin reflects bld glucse f even shrter term than fructsamine, because fr the cncentratin f glycated albumin t enter the nrmal reference interval, whether as a result f strict diet r intensive therapy with insulin, takes fur weeks (16). An index reflecting the cncentratin f bld glucse in the preceding week wuld be useful in the initial stage f treatment f type II diabetes as well as in evaluatins f the unstable phase in patients with severe diabetes. Yung erythrcytes have a lwer Hb A1,, cntent than d the lder cells (17, 18). New erythrcytes, released int the bldstream with little Hb A1,,, underg glycatin f hemglbin thrughut their life span as they are expsed t glucse in the plasma. The lder the cell and the higher the Department f Labratry Medicine, St. Luke s Cllege f Nursing and St. Luke s Internatinal Hspital, 1-1, Akashi-ch, Chuku, Tky 14, Japan. Received Octber 1, 1988; accepted March 13, cncentratin f plasma glucse, the greater the pprtunity fr this glycatin t ccur. We suggest that estimating Hb A1,, in erythrcytes f a specified age gives mre-useful specific retrspective infrmatin abut the glycemic status f a patient ver a preceding time interval. Hw lng a time interval is invlved depends n the age f the cells in which Jib A1,, is measured. In this study, we examined the relatinship f cell aging t glycatin, using an innvative methd fr erythrcyte fractinatin befre Jib A1,, assay. We examined the Hb A1 cncentratin in fractinated erythrcytes frm nrmal subjects and diabetic patients, and we cmpared these results with thse fr plasma fructsamine and whle-bld Hb A1 in the same specimens. Materials and Methds Bld was sampled frm tw grups f patients: knwn diabetic patients being mnitred at ur utpatient clinic and admitted t ur hspital after a 16-h fast, and nrmal fasting subjects wh were being admitted t the hspital fr health screening. This secnd grup began a 75-g ral glucse-tlerance test at the time f preprandial bld cllectin. Bld fr Jib A1,, and plasma fructsamine measurements was cllected frm the antecubital vein int a heparin-cntaining Vacutainer Tube (Bectn Dickinsn and C., Rutherfrd, NJ 77; cat. n. 493). Within 4 h, the bld specimens were manipulated fr erythrcyte fractinatin and separatin frm plasma (see belw). The plasma was stred at -8 #{176}C until fructsamine was measured. Plasma glucse was measured in an autmated glucse analyzer (Aut & Stat GA-112; Kyt Daiichi Kagaku C., Kyt 61, Japan) with use f glucse xidase (C ). All subjects were then classified int three grups accrding t the Wrld Health Organizatin s criteria (19): thse with nrmal glucse tlerance, thse with impaired glucse tlerance, and thse with diabetes mellitus. Reticulcytes were cunted in thin smears after staining with new methylene blue slutin accrding t the usual labratry methd (2). We measured pyruvate kinase (PK; BC ) activity in the fractinated erythrcytes by Beutler s methd (21). We measured Hb A1 with an autmated HPLC analyzer (Hi-Auth A1,, type 8121; Kyt Daiichi Kagaku C., Kyt 61, Japan) equipped with Aut-Sampler (type 9411) fr lading 1 samples at a time. An imprved mdel frm ne described earlier (22), this measurement in this analyzer takes nly 4 mm per sample. Measurement f plasna fructsamine was autmated by using a Cbas Mira centrifugal analyzer and a cmmercial kit frm Nippn-Rche (Chiyda-ku, Tky 1, Japan). Analyzer settings were as recmmended by the kit manufacturers. rythrcyte Fractinatin By heating, we sealed ne end f a plyprpylene tube (1.75 mm inner diameter, 2.95 mm uter diameter, 7. cm, identical t the tube used t hld ink fr a ballpint pen) 958 CLINICAL CHMISTRY, Vl. 35, N. 6, 1989

2 and fitted a small funnel-like adaptr, als plyprpylene, ver the ther end f the tube (Figure 1). After delivering.5 ml f heparinized bld int the adaptr, we centri- im tk ub& with th M ggg, c g 25 #{176}C. The adaptr-nw cntaining plasma, huffy cat, and an excess f erythrcytes-was cnnected t an aspiratr and simultaneusly remved frm the tube. We then further centrifuged the tube at x g fr 15 mm at 25#{176}Cwith a hematcrit centrifuge (Hematcrit KH-12S; Kubta, Bunkyu-ku, Tky 113, Japan), which had been adapted t hld the plyprpylene tube. After centrifugatin, we cut thrugh the tube at the brder between plasma and the packed erythrcytes, leaving a 5- t 6-cm-lng plyprpylene tube f densely packed erythrcytes. The heavier, lder cells were at the bttm; the lighter, newer cells were at the tp. We aspirated a 1.5-ML sample f erythrcytea frm the tp f the packed erythrcytes thrugh a 23-gauge venipuncture needle cnnected t an Authdiluter (Type 9211, Kyth Daiichi Kagaku). We bent the end f the needle, t limit its depth f insertin int the tube (Figure 1). We then cut a small segment with a length Vie that f the packed-cell tube with a small guilltine by fitting the cut end f the tube nt the stpper. The prcedure f aspirating the sample and cutting t discard the resulting small segments was repeated. The samples frm the 16 segments s btained were designated Fl (the lightest, yungest cells) t F16 (the heaviest, ldest cells). The packed cells are s viscus and the inner diameter f the tube is s small that the centrifuged packed cells are nt intermixed during the sampling frm the tube. We then transferred each sample t a sample cup, where it was diluted with.45 ml f hemlyzing slutin (8 mml/l phsphate buffer, ph 7.4, cntaining 1 g f Tritn X-1 per liter) by the autdilutr. We then placed the sample cups int the Aut-Sampler f the Hb A1,, analyzer. By radilabeling erythrcytes, Dann and Marikvsky (23) demnstrated that a mixture f phthalate esters facilitates the separatin f erythrcytes by age. Therefre, t determine whether phthalate ester must be added fr reliable separatin f the erythrcytes by age, we repeated the whle fractin prcedure, adding phthalate ester f knwn density [a mixture f dimethyl phthalate (relative density 1.189) and dibutyl phthalate (relative density 1.42)] t the tube befre and after the secnd centrifugatin, accrding t the methd described previusly (24). Results and Discussin Figure 3 shws reticulcyte cunts and PK activities. a). n aspiratr Fig. 1. Steps in the prcedure fr fractinating erythrcytes Reticulcytes were mst markedly numerus in Fl, mdi- II- / needle tube u be hlder handle blade / length f ne - mvable fractin FIg. 2. Hand-made cutter fr fractinating tube cntaining erythrcytes (viewed frm abve and In perspective) The stpper is mvable alng the axis shwn, Its psitin depending n the length f the tube and number f segments desired. If the length f the tube Is 6.cm and 16 segments are wanted, the cutting length (I) per segment Is 3.8 mm (6/16) - IcJ, > I1. In C Fractins Fig. 3. Mean pyruvate kinase activities () and retlculcyte cunts (S) f fractinated erythrcytes frm live nrmal subjects: three men (ages 48, 58, and 64 y) and tw wmen (age 37 and 71 y) Bars indicate ±SD I. -directin f mvement 8 CLINICAL CHMISTRY, Vl. 35, N. 6,

3 B A 6 11 Fractins Fig. 4. Hb A1 cntent f erythrcytes frm a nrmal subject (A) and a diabetic patient (B), fractinated by the present methd (#{149}) and by the methd invlving phthalate ester as separatrs () cating that the yungest cells were cntained in Fl. PK activity was greatest in Fl and declined linearly frm F2 t F16. Micrscpically, leukcytes were visible nly in Fl but nt in F2 t F16, mst having been remved with buffy cat in the adaptr. Because PK activity is greater in leukcytes than in erythrcytes, the markedly increased PK in Fl may have been caused by cntaminating leukcytes. Because the activity f PK in erythrcytes depends n their ages (25), its linear decrease f activity frm F2 t F16 verifies that the cells n each fractin were in fact separated accrding t their age. T determine Hb A1 in the erythrcytes frm a nrmal subject and a diabetic patient, we fractinated each samples by the present methd and by the methd invlving phthalate ester separatrs (24). Values fr Hb A1 in each fractin f the sample fractinated withut separatrs were essentially identical t thse f the sample fractinated with separatrs (Figure 4). Hwever, in the latter samples, sme fractins were unbtainable because f the presence f the separatrs: fractins 1 and 14 in the diabetic patient and fractins 6, 1, and 11 in the nrmal subject (Figure 4). Intra-assay cefficients f variatin (CVs) fr Hb A1 were.88% fr whle unfractinated erythrcytes (mean ± SD Hb A1: 5.67% ±.5% f ttal Hb), 2.43% in Fl (4.11% ±.1%),.88% in F6 (5.68% ±.5%), 1.22 in F11 (5.75% ±.7%), and.73% in Fl6 (5.45% ±.4%) (n = 2 each). This indicates that ur technique fractinates erythrcytes reprducibly accrding t their age, withut need fr any separatr r density liquid. This is pssible because living erythrcytes are very defrmable. Defrmable erythrcytes suspended in density liquid act as separable cells, but by themselves they are able t act as a density liquid. They are therefre able t arrange themselves int a ppulatin having the same density during high-speed centrifugatin. The methd f Fitzgibbns et al. (17) requires 3 ml f bld, the additin f density liquid (dextran slutin), a large centrifuge, 6 mm f centrifugatin, and wash-steps t remve density liquid frm the erythrcytes fr tw 16 erythrcyte fractinatins in each sample. Obviusly, erythrcyte fractinatin was greatly simplified in the present study. Figure 5 shws mean values fr Hb A1 fractinated erythrcytes and whle bld, and the mean cncentratin f fructsamine in plasma f nrmal subjects and uncntrlled diabetic patients. Hb A1 values fr fractinated erythrcytes and whle bld, and plasma fructsamine in newly diagnsed diabetes mellitus withut treatment, are als shwn in Figure 5. In nrmal subjects, Hb A1 increased steadily with cell aging, indicating the slw cnversin f Hb A t Hb A1,, with saturability (26,27), and a prbable slight reversibility (28). In the uncntrlled diabetic patients (whm we defined as having Hb A1,, >6.5% r fructsamine >3. mml/l), the mean Hb A1,, f fractinated erythrcytes was higher, but shwed a similar pattern. Newly diagnsed patients wh had nt started treatment shwed relatively higher Hb A1,, in Fl-F6, reflecting the recent high bld glucse cncentratin. On treatment, their values rapidly became nrmal. Crrelatins between the cncentratin f Hb A1,, f each fractin and plasma fructsamine, and Hb A1,, f whle bld, are shwn in Figure 6. The best crrelatin is seen fr the yunger middle-aged cells (F4-8), the prest fr the yungest and the ldest cells. This indicates that plasma fructsamine best reflects the retrspective pattern f plasma glucse cncentratin fr abut the preceding 2 days rather than mre recently (the past seven days) r lnger (1 days). The fact that there is abut an r =.8 crrelatin f plasma fructsamine with Hb A1,, in F9-F12 is t be expected, because fructsamine als reflects the glycated prtein f lnger life span such as glycated glb Fractins 16 A B I -S 3. e C Fig. 5. Means fr Hb A1,, in fractinated erythrcytes (fractins 1-16) and in whle bld (A) and plasma fructsamine (B) frm 34 nrmal subjects (#{149}) (31 men, three wmen; mean age 57 y, range y) and 55 patients with uncntrlled diabetes () (Hb A1,, >6.5% r fructsamine >3. mml/l; 48 men, seven wmen, mean age 62 y, range 4-79 y) (bars indicate ±SD), and in nine cases f newly diagnsed diabetes mellitus withut treatment: V: 51-year-ld wman, : 57-year-ld man, : 36-year-ld man, #{149}: 65-year-ld man, : 66-year-ld wman, V: 48-year-ld man, <): 54-year-ld man, A: 56-year-ld man, #{149}: 62-year-ld man CLINICAL CHMISTRY, Vl. 35, N. 6, 1989

4 ) t M 4. -I a 4. 3 S A I V IL 2 1.,..,_ C) A B Fractins Fig. 7. Hb A1,, in fractinated erythrcytes (fractins 1-16), and In whle bld (A) and plasma fructsamine (B) f selected subjects with impaired glucse tlerance Fractins :5-year-ld man, #{149}: 63-year-ld man, V: 67-year-ld man, :62-year-ld FIg. 6. CefficIents f crrelatin between Hb A1,, f the fractinated wman, S: 68-year-ld man, : 54-year-ld man erythrcytes and plasma fructsamine (#{149}) and between Hb A1,, f fractinated erythrcytes and whle bld () n 137. Nrmal sulects, 3/3 (male/female), mean age 56y (range 39-79); subjects with impaired glucse blerance: 28/3 (M/F), mean age 57 y (range y); patients with diabetes mellltus: 65/8 (MIF), mean age 62y (range 4-78 y) ulin as well as the shrter-lived glycated albumin (15). Hb A1,, f fractinated erythrcytes crrelated better with that in whle bld in the lder cells than it did with fructsamine; this crrelatin was prest in the yungest cells. Our results suggest that Hb A1,, f yung erythrcytes is a much mre specific and clinically useful index f recently changed bld glucse cncentratin than is fructsainine. The Hb A1,, cntent f yung cells shuld first be restred t nrmal if ne is t btain a curve acrss cell fractins f the erythrcyte ppulatin, i.e., t btain a nrmal value fr an Hb A1,, in whle bld, which is the ultimate gal f diabetic cntrl. Such a patient with nrmal Hb A1,, in whle bld wuld indicate that the subject s hemglbin had nt been expsed t highly increased cncentratins f bld glucse. This gal shuld be attainable in patients with nn-insulin-dependent diabetes mellitus, given prper treatment. In mst patients with insulin-dependent diabetes mellitus, Hb A1,, in whle bld is cnsidered a reliable index f glycemic cntrl status (6). Hwever, in the severe diabetics treated with insulin, we fund that Hb A1,, ffl-2 was nearly nrmal, whereas that f FlO-13 was markedly higher (see, e.g., the case shwn in Figure 4). In severe cases f insulin-dependent diabetes it is impssible t avid episdes f high plasma glucse, s erythrcytes inevitably are expsed ccasinally t excess glucse, even when diabetic cntrl is gd (as assessed by mnitring the daily prfile f bld glucse). As Figure 4 clearly shws, measurement f Hb A1,, in middle-aged r lder cells (and therefre f whle-bld Hb A1,,), t determine insulin dse, wuld result in dangerus hypglycemia, as reprted previusly (4,8). In such cases, measuring the Hb A1,, in yung erythrcytes may be mre useful fr rapid mnitring at the first step f insulin therapy. Gals fr treatment in these cases must be develped fr each individual and culd be re-assessed frequently by separately measuring Hb A1,, in the yung and ld cells. In ur subjects with impaired glucse tlerance (i.e., their plasma glucse was g/l 12 mm after ingest- ing 75 g f glucse), a subgrup had nearly nrmal values fr Hb A1,, in their whle bld and nrmal fructsamine cncentratins in plasma, but higher prprtins f Hb A1,, in F1-16 (Figure 7). By aviding the use f yunger cells, Hb A1 f Fl-l6 might becme mre specific fr glycemic status in retrspective evaluatin. Althugh these subjects are nt diagnsed as having diabetes mellitus, their cases shuld be carefully fllwed as they are treated by diet adjustment. Fructsamine cncentratin depends n t many variables (the methd f estimatin measures nnspecific reducing substances as well as glycated albumin and glbulin) t be f specific value in mnitring glycemic cntrl (14,15). Hb A1,, f whle bld reflects nly a mean value fr glycated hemglbin acrss the whle erythrcyte ppulatin, whereas the Hb A1,, f new erythrcytes reflects glucse cncentratins during the preceding week, and Hb A1,, f the lder middle-aged erythrcyte ppulatin crrelates well with the lnger-term bld glucse cncentratin. In ur experience, fractinatin f erythrcytes by age can be dne reliably with small samples f whle bld when the described adaptatin f currently available apparatus is used. We anticipate further simplificatin f this methd by mdifying the Aut-Sampler t take Fl and FlO directly frm the centrifuge tube after ne centrifugatin. This wrk was supprted by grant n frm the Research Fundatin fr Health Science f St. Luke s Internatinal Hspital. References 1. Kenig RJ, Petersn CM, Jnes RL, Saudek C, Lehrman M, Cerami A. Crrelatin f glucse regulatin and hemglbin Aic in diabetes mellitus. N ngi J Med : Gnen B, Rubenstein All, Rchman H, Tanega SP, Hrwitz DL. Haemglbin A1: an indicatr f the metablic cntrl f diabetic patients. Lancet 1977;ii: Jvanvic L, Petersn CM. The clinical utility f glycsylated hemglbin. Am J Med 1981;7: Gldstein D, Parker KM, ngland JD, et al. Clinical applicatin f glycsylated hemglbin measurements. Diabetes 1982;31(Suppl 3): Nathan DM, Singer D, Hurxthal K, GdsnJD. The clinical infrmatin value f the glycsylated hemglbin assay. N ngi J Med 1984;31: CLINICAL CHMISTRY, Vl. 35, N. 6,

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