Diabetologia 9 by Springer-Verlag 1977

Transcription

1 Diabetlgia 13, (177) Diabetlgia by Springer-Verlag 177 Serum High Density Lipprtein in Diabetic Patients M. F. L. Lpes-Virella, P.G. Stne, and J.A. Clwell Department f Medicine, ndcrinlgy, Metablism, and Nutritin Divisin, Medical University f Suth Carlina, and Veterans Administratin Hspital, Charlestn, Suth Carlina, USA Summary. The purpse f the present investigatin was the study f HDL lipprtein changes in patients with diabetes mellitus. The cmparisn was made between 40 nrmal and 10 diabetic subjects and the fllwing data was btained: relative HDL cncentratin (plyacrylamide gel electrphresis), HDL-chlesterl and aplipprtein A cncentratins. We fund significant decreases in HDL (18-28%) and HDL-chlesterl (31-40 rag/ 100 ml) in mst diabetics except in thse with nrmalized serum levels f glucse and lipids (34% and 50 rag/100 ml respectively). There was a statistically significant difference in HDL and HDL-chlesterl cncentratins between patients in the latter grup and ther diabetic patients. There was a negative crrelatin between HDL and HDL-chlesterl and serum glucse levels. N statistically significant difference was fund when aplipprtein A was cmpared in nrmal and diabetic subjects. Our results suggest that a deficient binding f chlesterl t apprtein A might be present in diabetes. Key wrds: High density lipprteins (HDL), HDLchlesterl, aplipprtein A, serum glucse, diabetes mellitus, lipprteins. It is generally accepted that diabetics may have increased serum lipids and lipprteins and a greater degree f athersclersis than nrmal individuals. Very few studies have been dne n the phsphlipid and chlesterl cntent f HDL in diabetic patients [1, 2]. These studies have been dne in a small number f uncntrlled diabetic patients with marked hyperlipaemia and have shwn a decrease in the HDL chlesterl and phsphlipid levels, with an increase in thse levels after cntrl f the diabetes with insulin therapy. Recently, there has been an upsurge f interest in the study f the rle f alpha lipprtein (HDL) in determining the risk f ischaemic heart disease. Glmset pstulated a rle f alpha lipprtein in clearing chlesterl frm tissues [3]. Miller and Miller [4] fund a crrelatin between a lw level f HDL and athersclertic disease. Rhads et al. [5] described a lwer cncentratin f HDL chlesterl levels in individuals with ischaemic heart disease. Recently Berg et al. [6] and Albers et al. [7] described a significantly lwer cncentratin f apprtein A-I in patients with crnary heart disease when cmpared with nrmal individuals. Special attentin t diabetic patients was lacking in these studies. The present study was undertaken with the purpse f determining whether similar changes culd be fund amng diabetic patients, amng whm the incidence f athersclertic heart disease is definitively increased [8]. Materials and Methds Serum Samples Serum was btained frm 10 male diabetics wh had been admitted t the hspital r wh were fllwed regularly in an utpatient clinic. The diagnsis f diabetes had been previusly established in these patients by the repeated demnstratin f fasting hyperglycaemia (> 125 mg/dl). In 3 f them, chemical diabetes was diagnsed using the criteria f Fajans and Cnn []. If diabetes had its nset befre age 20, a diagnsis f juvenile nset diabetes was

2 286 M. F. L. Lpes-Virella et al.: Lipprtein in Diabetic Patients Table 1. Main clinical features f the patients studied Number f patients Patients in each grup Type f diabetes Adult nset Juvenile nset Duratin f diabetes 0-5 years years 27 Mre than 10 years 38 Medicatin Insulin + diet 0 Oral agents + diet Diet alne 10 Weight Degree f besity Nrmal 87 Mderately bese b 14 Markedly bese ~ 8 Mean S. D. (lb) Percentage deviatin frm ideal bdy weight (xis) a Fr definitin f the grups refer t Table 2 b Between 15-25% abve ideal bdy weight c >25% abve ideal bdy weight Grup (2) (3) (4) (5) a made. If the nset was ver age 20, a diagnsis f maturity nset diabetes was made. Ninety-fur percent f the patients were n n ther medicatin except the ne prescribed fr diabetes cntrl. The remainder were als taking ant r antilipaemic medicatin. The mean age f the patient grup was 50.1 years and the range was frm years. The mean weight f the grup was lb and the range was frm lb. Serum samples frm 40 cntrl males with verified nrmal values f serum lipids, lipprteins and glucse were cllected. Thirty f the cntrls had n knwn disease and were under n medicatin. The remaining ten were patients hspitalized in the services f Urlgy and Geriatrics with the fllwing diseases: prstatic hypertrphy [4]; pulmnary tuberculsis [1], treated with Isniazid; chrnic brnchitis [2] treated with Tedral and aminphylline, femur and tibial fractures [2]; cataract [1]. The mean age f the cntrl grup was 3.0 and the range years. The mean weight f the cntrl grup was and the range 110 t 210 lb. All bld samples were cllected after an vernight fast. Lipprtein lectrphresis and HDL Determinatin Serum lipprteins were separated by cnventinal lipprtein plyacrylamide gel electrphresis using Sudan Black B as the lipid stain [10, 11]. The separatins were scanned with a Quick Scan Densitmeter and the relative cncentratin f HDL determined frm the integral values fr each fractin prvided by the scanner. Separatin f HDL HDL was separated by precipitatin f LDL and VLDL with sdium phsphtungstate in the presence f MgC12, as described by Burnstein [12, 13]. We added 100 ~1 f 4% sdium phsphtungstate slutin and 25 ~tl f 2M MgC12 t 1 ml f serum. This resulted in an immediate, cmplete and selective precipitatin f LDL and VLDL. Sdium phsphtungstate was used as a precipitating agent instead f heparin t avid the incmplete precipitatin that ccasinally ccurs with sme preparatins f heparin [14]. The supernatant was used fr determinatin f the chlesterl cntent f HDL. In highly lipaemic sera, if the initial precipitatin was nt cmplete (as judged by lipprtein electrphresis), the serum was diluted and the prcedure repeated t ensure a cmplete precipitatin. In rder t determine if the chlesterl determinatin by the methd used in ur labratry was affected by the presence f sdium phsphtungstate r magnesium, supernatants f 10 sera with variable chlesterl, triglycerides and glucse cncentratins

3 M. F. L. Lpes-Virella et al.: Lipprtein in Diabetic Patients were precipitated with 4% sdium phsphtungstate as described abve and with heparin (5000 U/ml) in the presence f 1M MnCI 2 [14]. We used 40 ~tl f heparin and 50 ~tl f 1 M MnC12 fr 1 ml f serum. Cmpleteness f precipitatin by bth methds was assessed by electrphresis. quivalent values f chlesterl were fund in the supernatants btained after precipitatin with bth precipitating agents. Quantitative Study f Serum Aplipprtein A (Ap A) Aplipprtein A was determined in nrmal individuals and diabetic subjects by radial immundiffusin [15]. Specific antisera t human alpha lipprtein prduced in rabbits was supplied by Behring Diagnstics (Lt 272 C, titre 0.1 U/ml). This antiserum has been characterized by Lee and Alaupvic and fund t cntain antibdies t A aplipprtein nly [16]. A standard f HDL prepared in ur labratry fllwing a methd described by Burnstein [12] was used. The purity f HDL standard was demnstrated by immunelectrphresis [17]. ach radial immundiffusin plate was prepared with 16 ml f 1% (w/v) agar-gel slutin; the antiserum cncentratin used in each plate was 4%. The sera samples were diluted t 1 : 10 and 1 : 20 in saline and 5 Ixl f the sample applied. ach serum sample was tested at least in duplicate in each f the abve dilutins. The immunlgical reactin was allwed t develp at rm temperature fr 24 t 48 h. In each plate we included 4 dilutins f the HDL standard as references, run in duplicate. The plates were washed with several changes f saline and finally rinsed vernight with distilled water and stained after drying with Cmassie brilliant blue R. Prtein Determinatin Prtein determinatin f the HDL standard was dne by the biuret reactin. Glucse Determinatin 287 Glucse levels were assayed using an autmated methd [20]. ~" Lipprtein Ultracentrifugal Separatin Venus bld samples were cllected in DTA (1 mg per ml f bld) frm 5 diabetic patients after a h fast. The plasma was separated by centrifugatin at 2500 rpm fr 20 min at 4 ~ C. Lipprtein separatin was initiated in the same day f cllectin, by sequential ultracentrifugatin. Ten ml f plasma were pipetted and verlaid with 0.16 M NaC1, ph, density g/ml. These samples were ultracentrifuged at rpm fr 24 h at 16 ~ C in a type 40 rtr in a Beckman Mdel L5-75 ultracentrifuge. The tubes were sliced at the middle f the clear zne separating the supra and infranatant. The bttm fractin was stirred and aspirated t a 10 ml vlumetric flask. The walls f the tube were carefully washed with saline and the sample adjusted t a final vlume f 10 ml. Five ml f that fractin cntaining LDL and HDL lipprteins were adjusted t a density f g/ml with slid NaC1 and run at rpm during 24 h at 16~ The tubes were sliced again in the middle f the clear zne separating the supra and infranatant and the bttm fractin was transferred t a vlumetric flask f 5 ml. Ttal plasma, HDL+LDL fractin and HDL fractin were used fr chlesterl and aplipprtein A assay as described. Statistical Methds Mean differences between grups were tested using the tw tailed t test. The crrelatin cefficient (r) was determined by linear regressin analysis. Statistical significance f the crrelatin cefficient was determined by the methd f Fisher and Yates. Chlesterl and Triglyceride Determinatins Ttal chlesterl was determined by an autmated clrimetric methd described by Blck et al after extractin with isprpanl [18]. The chlesterl cntent f HDL was assayed in a 1 : 10 isprpanl extract instead f the usual 1 : 20 extractin dne fr ttal sera. Triglycerides were determined by an autmated flurimetric methd [1]. Results We included 14 individuals in the present studies, divided int ne grup f nrmal subjects and fur grups f diabetic patients. Table 1 shws a summary f clinical data. In Table 2 ur labratry studies are summarized. Diabetic patients have lw levels f HDL and HDL-chlesterl. These findings are seen mainly in

4 288 M.F.L. Lpes-Virella et al.: Lipprtein in Diabetic Patients Table 2. Serum lipids, glucse, HDL, HDL-chlesterl and aplipprtein A levels in nrmal and diabetic subjects N. f Serum Ttal Serum Serum Glucse HDL Levels Chlesterl Aplipprtein Subjects Chlesterl Triglycerides (Plyacrylamide Cntent f A Levels lectrphresis) HDL (mg/100 ml) (mg/100 ml) (mg/100 ml) Percentage Values (mg/100 ml) (mg/100 ml) Grup a _+33 ( ) b (35-160) (55-108) (25-57) ( ) ( ) Grup c ~ ~ ( ) (30-135) (75-117) (23-55) (34-70) ( ) Grup d d c (160-24) (55-147) ( ) (16-42) (24--66) (121-23) Grup a 13+4U ( ) ( ) (7-123) (-33) (14-74) ( ) Grup d c (157-32) ( ) ( ) (7-35) (13-47) ( ) Grup 1 - Nrmal individuals Grup 2 - Diabetic subjects with nrmal serum lipid and glucse levels <125 mg/100 ml Grup 3 - Diabetic subjects with nrmal serum lipid and increased glucse levels Grup 4 - Diabetic subjects with increased serum lipid (chlesterl and/r triglycerides) and nrmal glucse levels Grup 5 - Diabetic subjects with increased serum lipid (chlesterl and/r triglycerides) and glucse levels a mean and standard deviatin b range c N. S. (as cmpared t Grup 1) d p<0.001 (as cmpared t Grup 1) Table 3. Chlesterl and Ap A levels in ttal plasma and ultracentrifugal lipprtein fractins Patient Glucse Trigl. Ttal HDL VLDL LDL Ttal HDL Chl. Chl. Chl. Chl. Ap A Ap A rng/100 ml mg/100 ml mg/100 ml mg/100 ml mg/100 ml rag/100 ml mg/100 ml mg/100 ml LDL + HDL Ap A mg/100 ml Grup 2 H.L B.V Grup 3 G.B L.B Grup 5 H.M Table 4. Age-related variatin f HDL and HDL chlesterl in cntrls Age Number HDL HDL f Chlesterl percentage subjects mg/100 ml (% f ttal lipid) yearsl years " 3+7 a 40-50years a 40+5 a 50-60years a 38+8 ~ 60-82years 5 51_+ a 3+3 a a N.S. (as cmpared with the grup with ages between years) the grup f hyperglycaemic and hyperlipidaemic patients (Grups 3-5). The levels f aplipprtein A d nt shw significant differences amng the several grups f individuals included in the study. The differences in the prprtin f HDL lipprteins and in the cncentratin f HDL-chlesterl amng the different grups were statistically analyzed. There were significant differences amng bth determinatins when nrmals (Grup 1) were cmpared with diabetics with elevated serum glucse and/r lipid values (Grups 3, 4, and 5) while there was n difference between nrmals and diabetics with nrmal serum glucse and lipid levels (Grup 2).

5 M. F. L. Lpes-Virella et al.: Lipprtein in Diabetic Patients NORMAL INDIVIDUALS c~ ,=::. ~ ~ 0 ~ ~ g~ =3 0 DIABTIC SUBJCTS WfTH NORMALIZD LVLS OF GLUCOS AND LiRIDS DIABTIC SUBJCTS WITH NORMALIZD A LVLS OF LIPIDS AND INCRASD GLUCOS LVLS L3 DIABTIC SUBJCTS WITH INCRASD LVLS OF LIPIDS AND GLUCOS z3 CO lo- 4' 8b z~ zs Serum Glucse (mg/looml) Fig. 1. Relatin between glucse cncentratins and the percentage value f HDL amng ttal lipprteins. The patients with nrmalized glucse and hyperlipprteinaemia (Grup 4) were excluded (n = 128, r = -0.50, p < 0.01) 80- NORMAL INDIVIDUALS DIABTIC SUBJCTS WITH NORMALIZD LVLS OF GLUCOS AND LIPlDS O O,% ", A 0 DIABTIC SUBJCTS WITH NORMALIZD LVLS OF LIPIDS AND INCRASD GLUCOS LVLS DIABTIC SUBJCTS WITH INCRASD LVLS OF LIPIDS AND GLUCOS ~ ~ ~ 40 ~ g~ 3 n- z~ 40 e i Serum Glucse (rng/looml) Fig. 2. Relatin between glucse cncentratins and HDL chlesterl levels. The patients with nrmalized glucse and hyperlipprteinaemia (Grup 4) were excluded (n , r = -0.51, p < 0.01) Statistically significant differences were als fund when the grup f well cntrlled diabetics (Grup 2) was cmpared with the ther grups f diabetics (3, 4, and 5). This was particularly striking in the cmparisn f Grups 2 and 5. Statistical analysis cnfirmed the apparent lack f a significant difference between the apprtein A cncentratins in the several grups f patients and nrmal individuals. In view f the nrmal levels f Ap A in diabetics with lw levels f HDL chlesterl, we perfrmed experiments t determine if, in these patients, there was a shift f Ap A frm HDL t anther lipprtein fractin mainly VLDL. In Table 3 ur results are summarized. It is apparent that there is n alteratin in the ttal amunt f Ap A in each ne f the fractins separated by ultracentrifugatin. As in nrmals mst f the Ap

6 20 M. F. L. Lpes-Virella et al.: Lipprtein in Diabetic Patients A was in the HDL fractin. The small differences that we fund are due t the nrmal variatin f the methd used fr Ap A assay. The crrelatins between the several variables studied were further explred by linear regressin analysis cmparing glucse cncentratins with a) percentage value f HDL amng ttal lipprteins, b) HDL-chlesterl, and c) aplipprtein A cncentratins. Sme f these studies are graphically illustrated in Figures 1 and 2. There were negative crrelatins between HDL and HDL-chlesterl and glucse levels, but n crrelatin was apparent when aplipprtein A and glucse cncentratins were cmpared. Furteen f the patients were mderately bese. They were distributed in grups 2, 3, 4 and 5 and their values fr HDL chlesterl and HDL percentage were nt significantly different frm the mean values fr the nn-bese members f the same grup. Amng the eight patients with marked besity, six f them were in Grup 5. There was n significant difference between the mean values fr HDL and HDL-chlesterl fr these individuals (15% and 2 mg/100 ml) and the nn-bese members f Grup 5 (18% and 31 mg/100 ml). The ther tw markedly bese individuals were in Grups 3 and 4. In bth cases, the values fr HDL (24.8% and 18.7%) and HDL-chlesterl (28 and 22.5 mg/100ml) were markedly belw the mean values fr the nn-bese members f the grup. Discussin This study demnstrates that male diabetic patients have abnrmalities in their HDL-lipprtein system, cnsisting f a decrease f the percentage value f HDL amng ttal lipprteins and in a decrease f HDL-chlesterl. On the cntrary, aplipprtein A cncentratins were nt significantly changed. An interesting crrelatin between ther indices f metablic dysfunctin and HDL abnrmalities exists. Well cntrlled diabetics with nrmal glucse and nrmal serum lipid levels have nrmal HDL values, while thse patients with nrmal serum lipids, but with abnrmal glucse cncentratins, shw abnrmal HDL values. Furthermre, linear regressin analysis demnstrated a crrelatin between glucse cncentratin, HDL percentage and HDL-chlesterl. This finding can be significant cnsidering the evidence that has been recently accumulated suggesting that lw alpha chlesterl values are assciated with athersclertic crnary disease [4, 5]. The increased incidence f athersclertic disease in diabetics culd then be related t the pstulated clearing actin f alpha lipprtein n tissue chlesterl. Recent studies have als shwn a crrelatin between HDL chlesterl levels and aplipprtein A-I values [7]. This was nt the case in ur study, but we used an antiserum that wuld recgnize bth plypeptides f apprtein A (ap A-I and A-II). It is knwn that the rati f Ap I t Ap II is very imprtant in determining the binding capacity f these plypeptides t HDL lipids [21]. It is pssible that ur lack f crrelatin between Ap A cncentratins and HDL chlesterl levels results frm an imbalance f Ap I and Ap II cncentratins that smehw wuld result in a decreased binding capacity fr chlesterl. This is a pint that deserves further investigatin. Alternatively, a pssible cnsequence f the metablic changes ccurring in diabetic patients is that the availability f chlesterl t apprtein A decreases. One pssible criticism f ur study is the age difference between the grup f nrmal individuals and the patient grup. Hwever, Berg et al. [6] have reprted that HDL cncentratins, althugh sex-dependent, d nt vary significantly with age. HDLchlesterl has als been fund nt t vary with age [6] r alternatively, t increase with increasing age [14]. In ur studies, we saw n age-related changes f HDL r HDL-chlesterl in the cntrl grup (Table 4): Further, in spite f the fact that the ages did nt differ significantly in the 4 diabetic grups (Grup 2: ; Grup 3: ; Grup 4: ; and Grup 5: ), we saw significant differences in HDL and HDL-chlesterl when Grup 2 was cmpared with the thers. There des nt appear t be a majr influence f besity n HDL r HDL-chlesterl levels in ur studies. Occasinally, hwever, the presence f marked besity was assciated with unusually lw levels f HDL and HDL chlesterl. This bservatin requires further study. We cnclude that HDL and HDL-chlesterl levels may be a very critical index f metablic nrmalizatil~ in diabetics. They prvide a better evaluatin than ttal lipid determinatins, since we have fund that hyperglycaemic and nrmlipaemic patients have significantly lwer values fr bth HDL and HDL-chlesterl. Finally, the implicatins f these findings regarding precise cntrl f bld glucse and its pssible influence n vascular disease in diabetes deserves cmment. In view f the fact that HDL-chlesterl and percentage is nly nrmalized at plasma glucse levels f abut 125 mg/100 ml r belw (Figs. 1 and

7 M. F. L. Lpes-Virella et al.: Lipprtein in Diabetic Patients 21 2), a strng argument fr diabetic management t nrmglycaemia can be made. If the pstulate f Miller et al that lwered alpha lipprtein relates t chlesterl clearing frm tissues is crrect, then nrmalizatin f this value in diabetics, a grup highly susceptible t athersclersis, culd be critical in the preventin f athersclersis. Acknwledgements. We wish t recgnize the valuable technical assistance prvided by Mrs. Julia McNeil and J.S. llis and t thank Ms. Dnna Dix and Janice Lane fr their excellent wrk in typing the manuscript. References 1. Furman, R.H., Hward, R.P., Jakshimi, K., Nrcia, L.N.: The serum lipids and lipprteins in nrmal and hyperlipidemie subjects as determined by preparative ultracentrifugatin. Am. J. Clln. Nutr., (161) 2. Crnwell, D.G., Kruger, F.A., Hamwi, G.J., Brwn, J.B.: Studies n the characterizatin f human serum lipprteins separated by ultracentrifugatin in a density gradient. Am. J. Clin. Nutr., (161) 3. Glmset, J.A.: The plasma lecithin-chlesterl acyltransferase reactin. J. Lipid Res., (168) 4. Miller, G.J., Miller, N..: Plasma high-density lipprtein cncentratin and develpment f ischaemic heart disease. Lancet 175 I, Rhads, G.G., Gnlbrandsen, C.L., Kagen, A.: Serum lipprteins and crnary heart disease in a ppulatin study f Hawaii Japanese men. New ngl. J. Med. 24, (176) 6, Berg, K,, Brresen, A.L., Dahlen, G.: Serum high density lipprtein and athersclertic heart disease. Lancet 176 I, Albers, J.J., Wahe, P.W., Cabana, V.G., Hazzard, W.R., Hver, J.J.: Quantitatin f Aplipprtein A-I f human plasma high density lipprtein. Metablism 25, (176) 8. Garcia, M.J., McNamara, P.M., Grdn, T., Kannell, W.B.: Mrbidity and mrtality in diabetics in the Framingham ppulatin. Diabetes 23, (174). Fajans, S.S., Cnn, J.W.: Apprach t a predictin f diabetes mellitus by mdificatin f glucse tlerance test with crtisne. Diabetes 3, (154) 10. Masker, B.H., Levy, R.I., Fredricksn, D.S.: The use f plyacrylamide gel electrphresis in differentiating type III hyperlipprteinemia. J. Lab Clin. Med. 81, (173) 11. Lpes-Virella, M.F., Virella, G., Parreira, F., Sequerra-Amram, S.: Analises cmparativ de algunas technicas para la separacin de las lipprteinas sericas. Rev. Clin. sp. 133, (174) 12. Burnstein, M.: Islement des lipprteines seriques de faible densite apres flculatin par le phsphtungstate de sude a ph neutre en presence de chlrure de magnesium. Nuv. Rev. Fr, Hematl. 3, (163) 13. Burnstein, M., Schlnick, H.F., Mrfin, R.: Rapid methd fr the islatin f lipprteins frm human serum by precipitatin with plyanins. J. Lipid Res. 11, (170) 14. Manual f lipid peratins. Lipid research clinics prgram. Vl. I. Lipid and lipprtein analysis. Department f Health ducatin and Welfare. Publicatin N.(NIH) (174) 15. Mancini, G., Carbnara, A.O., Heremans, J.F.: Immunchemical quantitatin f antigens by single radial immundiffusin. Immunchemistry 2, (165) 16. Lee,.M., Alaupvic, P.: Studies f the cmpsitin and structure f plasma lipprteins. Islatin, cmpsitin and immunchemical characterizatin f lw density lipprtein subfractins f human plasma. Bichemistry, (170) 17. Grabar, P., Williams, C.A.: Methde permettant l'etude cnjuguee des prprietes electrphretiques et immunchimiques d'un melange de prteines. Applicatin au serum sanguin. Bichim. Biphys. Acta 10, (153) 18. Blck, W.D., Jarrett, K.J., Jr., Levine, J.B.: An imprved autmated determinatin f serum ttal chlesterl with a single clr reagent. Cfin. Chem. 12, (166) 1. Kessler, G., Lederer: Flurmetric measurement f triglycerides. In: Autmatin in analytical chemistry. (d.: Keggs L. T., Jr.) p New Yrk: Mediad Inc Hffman, W.S.: A rapid phtelectric methd fr the determinatin f glucse in bld and urine. J. Bil. Chem. 120, (137) 21. Mrrisett, J.D., Jacksn, R.L., Gtt, A.M., Jr.: Lipprteins: Structure and functin. Ann. Rev; Bichem. 44, (175) Received: Nvember 22, 176, and in revised frm: February 15, 177 Dr. Mafia F.L. Lpes-Virella Department f Medicine Medical University f Suth Carlina 80 Barre Street Charlestn, SC 2401, USA