Diabetologia. Originals. Plasma Beta-thromboglobulin in Diabetes Meilitus

Size: px

Start display at page:

Download "Diabetologia. Originals. Plasma Beta-thromboglobulin in Diabetes Meilitus"

Diana Carter
5 years ago
Views:

Diabetlgia 18, 353-357 (1980) Diabetlgia 9 by Springer-Verlag 1980 Originals Plasma Beta-thrmbglbulin in Diabetes Meilitus D. Q. Brsey, J. Dawes 1, D. M. Fr

1 Diabetlgia 18, (1980) Diabetlgia 9 by Springer-Verlag 1980 Originals Plasma Beta-thrmbglbulin in Diabetes Meilitus D. Q. Brsey, J. Dawes 1, D. M. Fraser, C. V. Prwse 2, R. A. ltn 3, and B. F. Clarke Diabetic and Dietetic Department, Ryal Infirmary f dinburgh, 1M. R. C. Immunassay Team, 2Suth-ast Sctland Reginal Bld Transfusin Service, and 3Medical Cmputing and Statistics Unit, University f dinburgh, dinburgh, Sctland Summary. Plasma beta-thrmbglbulin was measured, using the dinburgh radiimmunassay technique and anticagulant mixture (cntaining prstaglandin l) in 61 nrmal subjects, 67 diabetics with and 54 diabetics withut micrangipathic cmplicatins. Plasma beta-thrmbglbulin was significantly higher in the diabetic patients (p < 0.01) but there was n significant difference between the tw diabetic grups. Twenty-six nrmal subjects, 27 diabetics with and 39 diabetics withut cmplicatins were studied further by measuring beta-thrmbglbulin in fur different ways using tw different radiimmunassay techniques and tw anticagulant mixtures (with and withut prstaglandin l). The dinburgh assay gave a value 1.97 times that btained with the Amersham assay, and the dinburgh anticagulant a value 0.78 times that with the Amersham anticagulant. Beta-thrmbglbulin cncentratin in the meniscus layer was apprximately twice that in the middle layer. The lwer betathrmbglbulin values btained with the dinburgh anticagulant may result frm a different sampling technique r frm preventin f in vitr beta-thrmbglbulin release, after venepuncture, by prstaglandin l. Abnrmal platelet behaviur in diabetes was cnfirmed althugh its rle in the pathgenesis f micrangipathic cmplicatins remains unclear. Key wrds: Plasma beta-thrmbglbulin, radiimmunassay technique, prstaglandin 1 anticagulant, plasma layer, diabetic micrangipathy, diabetes mellitus. Despite intensive research the precise pathgenesis f the specific micrangipathic cmplicatins f diabetes mellitus remains bscure [1]. Hwever, it seems clear that multiple factrs are invlved and, amngst these, abnrmalities f platelet functin may be f cnsiderable imprtance [1-3]. In 1975 a platelet specific prtein released during platelet aggregatin was islated and named betathrmbglbulin (fltg) [4]. A highly sensitive radiimmunassay has been develped fr its measurement in bilgical fluids [5] and a cmmercial radiimmunassay kit (Thrmbglbulin RIA kit, Radichemical Centre, Amersham) is nw available. Althugh n bilgical functin has yet been ascribed t/3tg it has been shwn t be elevated in certain thrmbemblic states [6-9]. Recent studies f the relatinship between plasma fitg levels and micrangipathic cmplicatins f diabetes have prduced incnsistent results [10-12]. Tw technical differences in btaining the plasma sample were nted between the abve studies. The anticagulant mixture used in dinburgh [10] included prstaglanding 1 (a ptent platelet antiaggregating agent) whereas the subsequent Sheffield [11] and Oxfrd [12] studies used the Amersham kit frm which prstaglandin was mitted. This led t the suggestin that plasma fitg may be nrmal in mst diabetic patients, the recrded increases [11, 12] being the result f in vitr fitg release after venesectin, and that a ptential platelet hypersensitivity may exist in diabetes which is blcked in the presence f prstaglandin 1 [13]. The secnd variatin cncerns the fractin f plasma taken fr assay. After centrifugatin f the bld samples users f the Amersham kit are recmmended t remve the tp 0.5 ml f the plasma (meniscus layer) fr assay whereas with the dinburgh technique the same vlume is remved frm the middle layer. It has been fund that platelets may be trapped at the meniscus and result in an elevated/3tg cncentratin in this layer [14]. We have studied three grups f subjects (nndiabetic cntrls and diabetics with and withut mi X/80/0018/0353/$01.00

2 354 D.Q. Brsey et al.: Beta-thrmbglbulin in Diabetes Table!. Details f subjects in whm plasma fl-thrmbglbulin was measured using the dinburgh anticagulant and dinburgh assay Subjects Age Sex Duratin f Plasma fl-thrm- Mean _+ SD % Male diabetes Treatment bglbulin Median and range Median and (Years) Insulin Oral agents Diet alne range (ng/ml) Nrmal subjects 34 _ (n = 61) (15-120) Ttal diabetics 55 _ a (n = 121) (0-45) (11-923) Diabetics withut cmplicatins (n = 54) (0-37) (11-923) Diabetics with cmplicatins b (n = 67) (4-45) (13-488) a p < 0.01 when cmpared with cntrls b p nt significant when cmpared with uncmplicated diabetics Wilcxn Rank Sum test crangipathic cmplicatins) and measured fitg cncentratins, n simultaneus bld samples, in fur ways by using the different anticagulant and assay techniques available. Furthermre, we have assessed the cntributin f the meniscus layer t variatin between assays. Patients and Methds Three grups f subjects were studied; [1] 61 nrmal subjects; [2] 54 diabetic patients withut retinpathy and clinical r bichemical evidence f nephrpathy; [3] 67 diabetic patients with significant retinpathy f whm 16 had evidence f nephrpathy. Significant retinpathy was defined as greater than 4 micraneurysms per fundus. Nineteen patients had backgrund retinpathy f mild r mderate severity and 48 patients either very extensive backgrund retinpathy r prliferative retinpathy. The age, sex, duratin f diabetes and treatment f the patients are shwn in Table 1. Nne f the subjects studied were receiving medicatins knwn t interfere with platelet functin. Venus samples (2.7 ml) were withdrawn withut stasis int plyprpylene syringes, and transferred immediately t a silicnised glass tube cntaining the platelet release inhibitrs edetic acid (DTA), prstaglandin 1 and thephylline (dinburgh anticagulant). The tubes were centrifuged at 1900 g fr ne hur at 0~ as previusly described [5]. The middle third f the platelet pr plasma was remved fr radiimmunassay f fltg as described by Bltn et al. (dinburgh assay) [15]. In all cases simultaneus venus plasma glucse cncentratins were estimated and in 46 diabetics ttal glycsylated haemglbin (HbA1) was als measured [16]. Ninety-tw patients (26 nrmal subjects, 39 diabetics withut and 27 diabetics with cmplicatins) were studied further by measuring fltg levels n simultaneus venus bld samples by fur different techniques; [1] dinburgh anticagulant and dinburgh assay, [2] dinburgh anticagulant and Amersham radiimmunassay kit, [3] Amersham anticagulant (DTA and thephylline but n prstaglandin 1 in a plastic tube) and dinburgh assay, [4] Amersham anticagulant and Amersham assay. In additin, bld samples were taken int Amersham tubes frm 24 cntrls and 24 diabetic patients. After centrifugatin f the sample, 0.1 ml f the meniscus layer, 0.5 ml f the middle layer and 0.1 ml f the deep liquid layer were taken fr fltg assay by the dinburgh methd. Statistical Methds The measured variables did nt have a nrmal distributin and the data is therefre expressed as median and range. The significance f difference between grups was assessed by the Wilcxn Rank Sum test. An analysis f variance was carried ut n the lgarithms f the fur values measured by different methds n each f the 92 samples. Lgarithms were used because examinatin f the data measured by each pair f methds suggested that differences in measurement values between methds were likely t be prprtinal rather than independent f the values themselves. The differences in verall mean value fr the tw anticagulants and tw assays in this analysis were cnverted (by taking antilgs) t estimates f the multiplicative factrs by which the tw methds differ. Cnfidence limits were placed n these estimates. A similar analysis f variance was carried ut n the measurement f fltg cncentratins in the meniscus, middle and deep layers f the samples. Results The median and range f fitg cncentratins in the three grups studied using the dinburgh anticagulant and the dinburgh assay is shwn in Table 1 and the individual values are depicted in Figure 1. The plasma fltg cncentratins were significantly higher in the diabetic patients than in the nrmal subjects (p < 0.01). Hwever, there was n significant difference between the fitg levels f diabetics with and withut cmplicatins. There was n crrelatin between the fitg cncentratins and the age, sex,

3 D. Q. Brsey et al.: Beta-thrmbglbulin in Diabetes duratin f diabetes, treatment, simultaneus plasma glucse r HbA 1 levels. The median and range f fltg levels btained by the 4 different measurement techniques is shwn in Table 2. There was a significant difference in the fltg levels between nn-diabetic and diabetic grups by all 4 methds but again n significant difference between diabetics with and withut cmplicatins. The results f the analysis f variance n the 4 measured values in each f the 92 samples is shwn in Table 3. There was n significant interactin between assays, anticagulant and patient type (diabetic r nrmal). This indicates that the data is cnsistent with a mdel in which the assay and anticagulant used have independent multiplicative effects n the expected measurement values, these effects als being independent f patient type. The variance rati fr samples within patient types shws that a highly significant difference exists in fltg values between different patients f the same type as well as between diabetics and nrmal subjects. It was estimated that, n average, the dinburgh assay will give a value that is 1.97 times that fr the Amersham assay (95% cnfidence limits, 1.85 and 2.09) and that use f the dinburgh anticagulant will give a value that is 0.78 times that when the Amersham anticagulant is used (0.73 and 0.83). The fit t the data f these estimates is exemplified by the brken line in Figure 2 which shws the relatinship expected when the factr f 1.97 (a cnstant difference f n the lgarithmic scale) is applied t the data using the dinburgh anticagulant. '~ c 3 c_.s _s ' ~q cj g0 4O L I" 4-! 8 I" $ ! ~ ~137.1 r $ i Ib _L C0ntr0[s Diabetics withut Diabetics with cmplicatins cmp[icati0ns (n=61) (n=54) (n = 67) Fig, 1. Plasma fl-thrmbglbulin cncentratins (dinburgh anticagulant and dinburgh assay) in diabetic patients, with and withut cmplicatins, and nrmal subjects. The hrizntal bars represent median values Table 2. Plasma fl-thrmbglbulin cncentratins in diabetic patients and nrmal subjects measured by 4 different techniques (median and range ng/ml) Subjects dinburgh assay Amersham assay Nrmal subjects 35 (n = 26) (18-200) Ttal diabetics 55 (u=66) (16-923) Diabetics withut 51 "1 cmplicatins (16-923) (n=39) Diabetics with 61 cmplicatins (32-488) (n=27) Wilcxn Rank Sum test 43 / 30} (20-195) (<10-85) (10-88) p<0.001 p<0.001 p<0.005 p< ( ) (10-218) (10-218) dinburgh anticagulant Amersham anticagulant dinburgh anticagulant Amersham anticagulant ( ) (10-218) (10-218) NS NS NS NS (31-480) (10-114) (17-141)

4 356 u3 t3 _c 2 n r- - LIA 3 9 2;./O 9 ///// D. Q. Brsey et al.: Beta-thrmbglbulin in Diabetes Differences between the lgarithms f the fltg cncentratins in the meniscus, middle and deep layers f plasma samples frm 24 nrmal cntrls and 24 diabetic patients are shwn in Table 4. The meniscus layer yielded n average a/3tg value apprximately twice that btained frm the middle layer, and the diabetics were significantly mre variable than the nrmals in the prprtinal difference between these tw layers. The mean lg difference between the middle and deep layers, hwever, was nt significant. Discussin F I I Amersham assay Fig. 1. Plt f plasma fl-thrmbglbulin values btained using dinburgh anticagulant with the tw different assays (lgarithmic scale) fr nn-diabetic (9 and diabetic (O) patients. The slid line shws identity between the tw assays and the brken line the mean relatinship estimated frm the analysis f variance Table 3. Analysis f variance f the lgarithms f/3-thrmbglbulin as measured by fur methds n samples frm 66 diabetic patients and 26 nrmal subjects Surce f variatin Sum f Degrees f Mean Variance squares freedm variance rati Type f patient a Samples within types a Assay a Tube ~ Type assay Type tube Assay tube Type X assay tube Residual Ttal ap<0.001 The finding f elevated plasma fltg cncentratins in diabetics supprts the reprts frm Sheffield [11] and Oxfrd [12] althugh it cntrasts with a previus study in dinburgh [10] which shwed n significant difference in/3tg levels betwen nrmal subjects and diabetic patients with cmplicatins. This latter study is subject t statistical criticism. The difference in age between the diabetic patients (55 _+ 15 years) and the nrmal subjects (34 _+ 14 years) des nt accunt fr ur findings since Dewar et al. [17] fund n significant increase in plasma fltg with age thrughut the range we have studied. N significant difference was fund in fltg cncentratins between diabetics with and withut micrangipathic cmplicatins. This differs frm tw previus reprts [11, 12], althugh the Oxfrd wrkers fund that there was n crrelatin between the degree f fltg elevatin and the severity f cmplicatins (retinpathy, neurpathy and large vessel disease). By measuring/3tg by fur different techniques we have shwn that the anticagulant and the assay used have independent effects n the expected values. The lwer/3tg values btained using the dinburgh anticagulant may result frm preventin f in vitr/3tg release, after venepuncture, by the presence f prstaglandin l. Hwever, as the abve factrs are independent f patient type this must ccur in bth diabetics and nrmal subjects, rather than in diabetics alne as previusly suggested [13]. Table 4. Analysis f differences f the lgarithms f fl-thrmbglbulin between plasma layers frm 24 diabetic patients and 24 nrmal subjects Plasma layers Nrmal subjects Diabetics F t stimate and 95% Mean (SD) Mean (SD) cnfidence interval fr prprtinal difference Meniscus - middle 0.29 (0.15) 0.39 (0.23) 2.29 ~ (1.68, 2.85) Middle -deep 0.09 (0.18) 0.07 (0.18) (0.95, 1.53) Deep - meniscus (0.19) (0.26) (0.33, 0.60) p < 0.05

5 D. Q. Brsey et al.: Beta-thrmbglbulin in Diabetes 357 Perhaps mre imprtant is the effect f sampling frm the meniscus layer since this nt nly increases the /3TG cncentratin measured, but may als intrduce an extraneus surce f variatin between nrmal and patient samples. The lack f a significant difference in fltg values between the tw diabetic grups suggests that raised levels in diabetics are nt secndary t the develpment f micrangipathic cmplicatins. This is supprted by the finding f elevated/3tg levels in newly diagnsed insulin independent diabetics which fell nce plasma glucse cntrl was achieved [11]. This suggests that metablic disturbance may be respnsible fr the elevatin f/3tg in diabetics either by directly affecting functin r endthelial interactins f platelets. Althugh we fund n crrelatin between/3tg and simultaneus HbA1 and plasma glucse, fluctuatins in ther metablites such as lipids may result in elevated/3tg levels. Abnrmally high plasma chlesterl and triglyceride cncentratins have been reprted in tw uncmplicated diabetic patients with elevated/3tg levels (126 and 146 ng/ ml) [12]. Other pssible surces f raised/3tg cncentratins in diabetics include an increased platelet turnver, a primary platelet defect r vascular endthelial damage. Our study adds weight t the already cnsiderable evidence in favur f abnrmal platelet behaviur in diabetes. Hwever, definitive evidence fr the imprtance f abnrmal platelet functin in the pathgenesis f diabetic micrangipathy has yet t be prvided. Acknwledgements. We wish t thank Dr. J. D. Cash, Natinal Medical Directr f the Scttish Natinal Bld Transfusin Service and Dr. D. S. Pepper, Suth-ast Sctland Reginal Bld Transfusin Service, dinburgh, fr their expert advice. References 1. McMillan D, Ditzel J (1976) Prceedings f a Cnference n Diabetic Micrangipathy. Diabetes 25 [Suppl 2]: Mustard J F, Packham M A (1977) Platelets and diabetes mellitus. N ngl J Med 297: Waitzman MB, Clley AM, Nardelli-Olkwska K (1977) Metablic appraches t studies n diabetic micrangipathy. Diabetes 26: Mre S, Pepper D S, Cash J D (1975) The islatin and characterisatin f a platelet specific/3-glbulin (/3-thrmbglbulin) and the detectin f anti-urkinase and anti-plasmin released frm thrmbin-aggregated washed human platelets. Bichim Biphys Acta 379: Ludlam C A, Cash J D (1976) Studies n the liberatin f/3- thrmbglbulin frm human platelets in vitr. Br J Haematl 33: Ludlam C A, Bltn A, Mre S, Cash J D (1975) New rapid methd fr diagnsis f deep venus thrmbsis. Lancet II: Denham M J, Fisher M, James G, Hassan M (1977)/3-thrmbglbulin in clinical cnditins. Lancet I: Redman C WG, Allingtn M J, Bltn F G, Stirrat G M (1977) Plasma/3-thrmbglbulin in pre-eclampsia. Lancet II: Ludlam C A, Mre S, Bltn A, Pepper D S, Cash J D (1975) The release f a human platelet specific prtein measured by radiimmunassay. Thrmb Res 6: Campbell I W, Dawes J, Fraser D M, Pepper D S, Clarke B F, Duncan L J P, cash J D (1977) Plasma/3-thrmbglbulin in diabetes mellitus. Diabetes 26: Prestn F, Ward J D, Marcla B H, Prter N R, Timperley W R, O'Malley B C (1978) levated/3-thrmbglbulin levels and circulating platelet aggregates in diabetic micrangipathy. Lancet I: Burrws A W, Chavin S I, Hckaday T D R (1978) Plasmathrmbglbulin cncentratins in diabetes mellitus. Lancet I: ditrial (1978) Platelets, beta-thrmbglbulin and diabetes mellitus. Lancet I: Rasi V (1979) fl-thrmbglbulin in plasma; false high values caused by platelet enrichment f the tp layer f plasma during centrifugatin: Thrmb Res 15: Bltn A, Ludlam CA, Mre S, Pepper D S, Cash JD (1976) Three appraches t the radiimmnsassay f human fl-thrmbglbulin. Br J Haematl 33: Kynch PAM, Lehman H (1977) Rapid estimatin (21/2 hurs) f glycsylated haemglbin fr rutine purpses. Lancet II: Dewar H A, Marshall T, Weightman D, Prakash V, Bn P J (1979) Beta-thrmbglbulin in antecubital vein bld: the influence f age, sex and bld grup. Thrmb Haemstas 42: Fergusn JC, Mackay N, Philip JAD, Sumner DJ (1975) Determinatin f platelet and fibringen half-life with (75s) selenmethinine: studies in nrmal and in diabetic subjects. Clin Sci Ml Med 49: Received: June 25, 1979, and in revised frm: Nvember 23, 1979 Dr. D. Q. Brsey Diabetic and Dietetic Department Ryal Infirmary f dinburgh dinburgh H3 9YW Sctland

Obesity/Morbid Obesity/BMI

Obesity/Morbid Obesity/BMI Obesity/mrbid besity/bdy mass index (adult) Obesity/Mrbid Obesity/BMI Definitins and backgrund Diagnsis cde assignment is based n the prvider s clinical judgment and crrespnding medical recrd dcumentatin