Direct Assay of LDL Cholesterol

Transcription

1 CHEMISTRY Davd A. Armbruster, PhD, C(ASCP) Patrca A. Lambert, MS, MT(ASCP) Drect Assay of LDL Cholesterol Comparng Measurement and Calculaton Hypercholesterolema s one of the major rsk factors assocated wth coronary heart dsease (CHD). Although genetcs partly dctates a person's cholesterol level, det and medcaton also may have an effect. To assess an ndvdual's rsk of CHD and to treat patents wth hypercholesterolema, t s crucal that the values for the major serum lpds are accurate. These values nclude total cholesterol (TC), hgh-densty lpoproten cholesterol (HDL), low-densty lpoproten cholesterol (LDL), and trglycerdes. Total cholesterol s composed of HDL, LDL, ntermedate-densty lpoproten (IDL), very low-densty lpoproten (VLDL), and lpoproten (a) [Lp(a)].1 About 6% to 7% of total cholesterol s found n the LDL fracton. Low-densty lpoproten deposts cholesterol n tssue, creatng plaque that clogs coronary arteres; hgh LDL values are consstent wth ncreased CHD rsk. For ths reason, LDL s termed the "bad cholesterol." Hgh-densty lpoproten levels are nversely proportonal to CHD rsk; hgh HDL serum levels are thought to protect aganst CHD because HDL removes cholesterol from tssue and delvers t to the lver where t s metabolzed nto ble acds and ultmately excreted. Hgh-densty lpoproten therefore s called the "good cholesterol." Coronary heart dsease s a complex, multfactoral condton. Some rsk factors, such as gender, age, and genetc predsposton for developng CHD, cannot be modfed. Other rsk factors, such as hgh cholesterol levels, hypertenson, and cgarette smokng, can be modfed to decrease the rsk of CHD. The Natonal Cholesterol Educaton Program (NCEP) encourages everyone to "Know your number" your TC number, that s and to be aware of your CHD rsk, as llustrated n Table l. 2 Intal classfcaton of CHD rsk s based on screenng for TC, but LDL becomes the key for detary and drug treatment. ABSTRACT Low-densty lpoproten (LDL) cholesterol s a crtcal lpd subfracton because t s lnked drectly wth the rsk of coronary heart dsease (CHD). Current approaches to dagnosng and montorng CHD emphasze the sgnfcance of accurate, precse LDL values. Due to the nherent complexty of measurng LDL by the reference method, ultracentrfugaton, t usually s estmated by a calculaton that uses measured values for total cholesterol, hgh-densty lpoproten (HDL) cholesterol, and trglycerdes. Calculated LDL (CLDL) results may be naccurate, especally f a patent's trglycerde level s elevated, and t requres a specmen from a patent who has fasted. New assays allow for the drect measurement of LDL (DLDL) analogous to assays for HDL and do not requre the patent to fast. Whle CLDL typcally compares well wth ultracentrfugaton and DLDL, t may provde a result that s dfferent n some nstances; that dfference may have clncal sgnfcance. A method comparson study of CLDL and DLDL resulted n the followng lnear regresson equaton: DLDL = 1.67 CLDL , r =.974, n = 36. The NCEP's cut-pont numbers are, of necessty, oversmplfed. Acceptable, safe lpd values vary among ndvduals and should be nterpreted n lght of the total rsk factor pcture for every patent. The key lpd values, however, are a convenent way to educate the general publc about the relatonshp between lpds and CHD. The NCEP takes a conservatve approach and does not recommend nsttutng treatment to lower cholesterol levels on the bass of a sngle elevated value. Lpd analyses should be repeated to allow for the many varables that can cause elevatons. As the bologc varablty of LDL s about 9.5%, some nvestgators have recommended as From LTC Medcal Laboratores, Dallas. Reprnt requests to Dr Armbruster, 3541 Creeksde Court, Bedford, TX o 1

2 TABLE 1. NATIONAL CHOLESTEROL EDUCATION PROGRAM KEY LIPID VALUES FOR THE INITIAL CLASSIFICATION OF THE RISK OF CORONARY HEART DISEASE* Trglycerdes Interpretaton/Classfcaton Abnormally low level <34 Desrable level >35 <2 Desrable level Borderlne hgh level 2-4 Hgh level 4-1, Very hgh level >1, trglycerde level. Inaccurate LDL results can lead to the mscalculaton of CHD rsk. An ndvdual wth a falsely elevated LDL value may be gven unnecessary lp dlowerng therapy, or an ndvdual wth falsely low LDL may not be started on a 2-4 Borderlne hgh-rsk CHD therapeutc regmen to 4-1, Hgh-rsk CHD >16 >24 lower the rsk of CHD. *AII values are expressed as mg/dl. TC ndcates total cholesterol; LDL, low-densty lpoproten Recently, drect LDL cholesterol; HDL, hgh-densty lpoproten cholesterol; CHD, coronary heart dsease. (DLDL, for short) many as four LDL determnatons before makng methods have been ntroduced. An apolpoproten treatment decsons.3 Repeatedly hgh LDL values antbody separates the LDL from other lpd fracndcate the need to ntate detary or drug ther- tons. Once solated, LDL cholesterol s measured usng a routne TC method. Drect LDL does not apy to lower blood cholesterol.2'4 requre a specmen from a fastng patent, and t corlow-densty lpoproten s measured by three relates well wth the ultracentrfugaton reference procedure. We compared the tradtonal CLDL methods; wth a DLDL method for specmens submtted to 1. Ultracentrfugaton our laboratory for lpd profle analyss. 2. Calculaton 3. Drect assay Low-rsk CHD <2 <13 Although ultracentrfugaton ((3 quantfcaton) s the reference method, t s mpractcal for routne clncal use owng to ts complexty, the tme requred (18 hours at 15, X g), and the need for specal equpment. Clncal laboratores typcally use the Fredewald formula to calculate an LDL value (CLDL, for short).5 Calculated LDL s derved from the measured values of TC, HDL, and trglycerdes as follows: LDL = TC - [(HDL + trglycerdes)^] The amount of VLDL roughly equals the trglycerdes dvded by 5. DeLong and colleagues developed an alternate calculaton to correct for hgh trglycerde levels; the calculaton dvdes the trglycerde concentraton by 6.6 Usually, a CLDL value s relable and clncally useful, and the calculaton of LDL s an acceptable and establshed practce. A CLDL value may lack precson and accuracy, however; an excessve mprecson and bas s assocated wth each of the measured lpd values. The relatonshp between VLDL and trglycerdes s nonlnear and starts to show problems n the 2 to 4 mg/dl range, fallng off at the hgher trglycerde levels. A hgh degree of varablty assocated wth CLDL makes t dffcult to detect small changes n LDL.7 Specmens also must be from patents who have fasted because recent meal ngeston affects the serum 614 VOLUME 27. NUMBER 9 Materals and Methods Total Cholesterol Assay The Boehrnger Mannhem (Indanapols) cholesterol/hp reagent was used followng the manufacturer's drectons. The kt s based on a sequental cholesterol esterase/cholesterol oxdase/peroxdase modfed Trnder method. The assay was performed usng a Htach 717 analyzer (Boehrnger Mannhem). Typcal dayto-day coeffcents of varaton (CVs) for ths assay range from 2.5% to 3.5%. Hgh-Densty Lpoproten Assay The Boehrnger Mannhem HDL cholesterol kt was used followng the manufacturer's drectons. The procedure uses phosphotungstate Mg +2 to precptate LDL and VLDL n a button after centrfugaton. Hgh-densty lpoproten n the supernatant s measured usng the TC assay descrbed above. Typcal day-to-day CVs for ths assay range from 3% to 4%. Trglycerdes Assay The Boehrnger Mannhem trglycerdes/gpo kt was used followng the manufacturer's drectons. Ths procedure uses a sequental lpase/glycerol knase/glycerol phosphate oxdase/peroxdase method. The assay was performed usng a Htach 717 analyzer. Typcal day-to-day CVs for ths assay range from 2.5% to 4.5%.

3 Calculated Low-Densty Lpoproten Calculated LDL was performed usng the TC, HDL, and trglycerdes values usng the Fredewald formula as descrbed earler Drect Low-Densty Lpoproten Assay The LDL cholesterol test, developed by Genzyme (Cambrdge, Mass) and dstrbuted by Sgma (St Lous), was used. The test features an affntypurfed goat polyclonal antbody, mmoblzed on polystyrene beads, that bnds HDL and VLDL, but not LDL. The assay separates LDL from the chylomcrons, VLDL, and HDL usng an apolpoproten antbody-based fltraton system. The assay combnes 2 LIL of reagent and 3 xl of serum n a plastc nner tube. The nner tube then s placed n the outer tube, and both are centrfuged for 5 mnutes. The nner tube, contanng the beads, s dscarded and the LDL fltrate n the bottom of the outer tube s tested for cholesterol usng the routne cholesterol procedure. Day-to-day CVs for low and hgh controls, mean values 92 and 193 mg/dl, are 1.8% and 3.8%, respectvely. 3 2 Q - "-.V 1-, CLDL (mg/dl) other cases (D-F), the agreement s reasonably good, but dfferences between 5% and 13% are noted, and some potental for clncal msnterpretaton exsts. Patent D had a CLDL of 116 but a DLDL of 131. Usng the NCEP LDL cut-pont of 13, there s a low rsk of CHD by CLDL but Method Comparson Patent specmens were assayed for TC, HDL, borderlne hgh rsk by DLDL. trglycerdes, CLDL, and DLDL. The CLDL and The dscrepances between CLDL and DLDL DLDL values were compared usng lnear regres- are llustrated best by the nne patents whose son analyss, wth CLDL desgnated as the refer- data were wthheld from the method comparson study (Table 3). Fve of the patents have ence method and DLDL as the test method. hypercholesterolema wth a potental hgh rsk for CHD, and four have values below 2 mg/dl. Specmens Specmens submtted from the facltes routnely Sx have borderlne hgh trglycerde values, three served by our laboratory were tested. Of a total of have hgh values, and one has a very hgh value. 45 specmens from 13 men (ages 28-81) and 32 Dfferences between CLDL and DLDL vary from women (ages 26-86), 9 patents' data were a low of 2% to a hgh of 26%. The most draexcluded from lnear regresson analyss due to matc case s patent 2, for whom t s mpossble hgh trglycerde values or dsparty between to obtan even a CLDL n ths case because the CLDL and DLDL results. Blood was collected n estmated value s mg/dl (due to the very serum separator vacutaner tubes. Specmens hgh trglycerde and very low HDL values), were shpped refrgerated and stored from 1 C to whereas the DLDL s 56 mg/dl. The dsparty between CLDL and DLDL values would result n 8 C n the laboratory untl testng. dfferent assessments of the rsk for CHD or for clncal decsons when montorng patents on Results and Dscusson The lnear regresson plot of CLDL vs DLDL s detary and/or drug therapy for the purpose of shown n the Fgure. Calculated LDL and DLDL lowerng cholesterol levels. Note that 9 (2%) of compare well for ths set of patents, wth CLDL the patents n ths small study populaton values about 1.6 mg/dl lower than DLDL values. showed consderable dfferences between CLDL The data ponts ncluded patents wth trglyc- and DLDL. The hgh percentage of dscrepances erde values up to 335 mg/dl; values from lkely reflects the makeup of the study group patents above 4 mg/dl trglycerdes were prmarly elderly patents wth a documented or excluded. Table 2 shows that for some ndvduals suspected pathologc condton that resulted n a (A-C), CLDL and DLDL values are nearly dent- request for a complete lpd profle. Fewer dffercal wth less than 5% dfference between them. In ences would be expected n a younger, healthy populaton. Lnear regresson plot of CLDL vs DLDL. The lnear regresson equaton s: DLDL =1.67 CLDL , r =.974, n = 36. CLDL ndcates calculated low-densty lpoproten; DLDL, drect low-densty lpoproten. I Io c! 61S

4 TABLE 2. LIPID VALUES FOR SELECTED PATIENT SPECIMENS USED IN THE METHOD COMPARISON STUDY* TC Trglycerdes HDL CLDL DLDL A B C D LLI Patent F *AII values are expressed as mg/dl. TC ndcates total cholesterol; HDL, hgh-densty lpoproten; CLDL, calculated low-densty lpoproten; DLDL, drect low-densty lpoproten. Other workers also have evaluated the Sgma DLDL assay. 8 " 11 McNamara and colleagues compared the DLDL assay wth ultracentrfugaton. 8 Correlaton between the assays was very good (r =.97, n = 1), wth a slght negatve bas for normal subjects and a small postve bas for hypertrglycerdemc patents. Between-run CVs ranged from 2.% to 5.1%. Profcent laboratores that mantan a hgh degree of precson for ther TC, HDL, and trglycerde assays may generate CLDL wth a varablty of less than 5%. Coupled wth a bologc varablty of about 6% to 1%, total varablty of CLDL values s probably n the range of at least 11%. 12 To montor the effcency of cholesterol-reducng therapeutc regmens va LDL, a method should have a CV of less than 5%. Calculated LDL values may vary from 11% to 26%, whereas the drect LDL can attan a CV of less than 4% and s much preferable for detectng decreases n LDL n response to therapy.12 Ransdell and colleagues found day-to-day CVs to range from 3.2% to 6.2% and good correlaton wth the CLDL for specmens less than 4 mg/dl trglycerdes (n = 42, slope = 1.67, r =.912), but the correlaton for specmens greater than 4 mg/dl trglycerdes was unmpressve (n = 33, slope =.22, r =.332).9 Sngh and colleagues found DLDL day-to-day precson to be 3% and correlaton wth specmens from 42 to 4 mg/dl trglycerdes to be acceptable (n = 142, slope =.9, r =.88), wth a lnear range of 25 to 27 mg/dl.1 Castellan and Ghadal also found that the DLDL correlates well wth the CLDL for specmens wth less than 4 mg/dl trglycerdes (slope =.986, r=.966). u The Genzyme assay marketed by Sgma and evaluated here s also avalable from Ponte Scentfc (Lncoln Park, Mch). Performance of the Ponte assay should be smlar to the Sgma product. We antcpate that other manufacturers 616 wll offer mmunoassay-based procedures for LDL n the future. Helena Laboratores (Beaumont, Tex) offers an electrophoretc assay for DLDL, based on the electrophoretc, rather than mmunosorbent, separaton of the lpd subfractons. Although the automated procedure smultaneously measures HDL and LDL, t s necessary to perform a chemcal measurement of total cholesterol to calculate the amount of HDL and LDL usng the electrophoretc data. Calculated LDL s used routnely and provdes a useful estmate of LDL for most patents. About 9% of CLDL values from typcal patents are wthn about 1% of ultracentrfugaton values.13 Calculated LDL stll can be used relably for most ndvduals whose trglycerde concentraton s less than 4 mg/dl. The DLDL assays may be useful n some cases because they offer greater precson and accuracy than CLDL, are not affected by elevated trglycerdes, and do not requre a fastng specmen. Cole and Ferguson reported that the Sgma DLDL test s unaffected by trglycerde concentratons up to 1,1 mg/dl.14 After a patent s dentfed as hypercholesterolemc and lpd-lowerng therapy s nsttuted, the success of the therapeutc regmen may be montored by LDL values alone. The NCEP recommends measurng LDL every 4 months durng the frst 18 months of therapy and then bannually. Drawbacks to DLDL testng nclude the relatvely hgh reagent cost (CLDL costs nothng because t s a calculated value), addtonal labor and tme to perform the assay, and potental problems wth rembursement from Medcare (CLDL has not been rembursed by Medcare snce 1992). Calculated LDL values depend on the accuracy and precson of the three ndvdual lpd assays requred for the calculaton, requre a fastng specmen, and are not recommended n the presence of chylomcrons, when trglycerdes exceed 4 mg/dl, or for patents wth Type III hyperlpoprotenema. 1 McNamara and colleagues found that even when trglycerdes are less than 2 mg/dl, about 14% to 16% of the populaton may be potentally msclassfed on the bass of LDL when usng the Fredewald formula. 7 Between 3 and 4 mg/dl trglycerdes, 41% of specmens tested devated by greater than 1% from ultracentrfugaton values and above 4 mg/dl, more than 57% of CLDL values dsagreed by greater than 1%. The CLDL s most prone to msclassfcaton for ndvduals close to the NCEP cut-ponts and persons who have not fasted 12 to 14 hours. A rule of thumb suggests that a

5 1% decrease n LDL decreases the ncdence of CHD by 2%.15 Another measure of CHD rsk s the LDL to HDL rato, another reason for usng the more accurate DLDL nstead of CLDL.16 The problem of semantcs rased by the new DLDL assays should be recognzed. Unless specfcally clarfed, LDL may refer to ether a calculated LDL or a drect LDL. Here, we have used CLDL and DLDL as unambguous abbrevatons for the two types of LDL. Laboratores should specfy how LDL values are derved to avod confuson. Some laboratores may report both CLDL and DLDL n accordance wth clents' requests or nternal testng polces. For example, f the TC, HDL, and trglycerde values are normal, a CLDL may routnely be reported unless the clent specfcally asks for a DLDL. If one of the lpd values s abnormal, a DLDL may automatcally be performed as a reflex test. The LDL stuaton s now smlar to that for free thyroxne (FT4). A varety of drect FT4 assays now are avalable, and many endocrnologsts recommend a FT4 and senstve thyrod-stmulatng hormone (TSH) for thyrod screenng. FT4 also may refer to a calculated estmate of the FT4 concentraton, that s, the free thyroxne ndex (FTI) derved from the T uptake and total T4 values. Although both the CLDL and FTI are estmatons calculated from measured values, they may be totally acceptable for dagnoss and montorng of patents. The debate about when to measure LDL drectly as opposed to calculatng t most lkely wll contnue. Concluson Drect LDL assays represent an mportant advance n the ablty to provde accurate and precse values for a sgnfcant lpd subfracton. Drect LDL assays wll lkely be routnely used when analyzng specmens from ndvduals who have been dagnosed wth coronary heart dsease or for whom a CLDL value cannot be calculated due to elevated trglycerdes. Low-densty lpoproten values wll probably contnue to be calculated for most patents as they are typcally vald estmates, provdng that the assumptons mplct n the Fredewald equaton hold, and they requre no addtonal effort or expense. References 1. Bachork PS, Ross JW. Natonal Cholesterol Educaton Program recommendatons for measurement of low-densty lpoproten cholesterol: Executve summary. Cln Chem. 1995;41: Expert Panel. Report of the Natonal Cholesterol Educaton Program expert panel on detecton, evaluaton, and treatment of hgh blood cholesterol n adults. Arch Intern Med. 1988;148: Smth SJ, Cooper GR, Myers GL, Sampson EJ. Bologcal TABLE 3. LIPID VALUES FOR PATIENT SPECIMENS THAT WERE NOT INCLUDED IN THE METHOD COMPARISON STUDY * Patent TC Trglycerdes HDL CLDL DLDL (-12) , Patents' specmens were not ncluded due to elevated trglycerdes and/or wdely dsparate CLDL and DLDL values. All values are expressed as mg/dl. FC ndcates total cholesterol; HDL, hgh-densty lpoproten; CLDL, calculated low-densty lpoproten; DLDL, drect low-densty lpoproten. varablty n concentratons of serum lpds. Sources of varaton among results from publshed studes and composte predcted values. Cln Chem. 1993;39: Expert Panel. Summary of the second report of the Natonal Cholesterol Educaton Program (NCEP) expert panel on detecton, evaluaton, and treatment of hgh blood cholesterol n adults (Adult Treatment Panel II). JAMA. 1993; 269: Fredewald WT, Levy RI, Frederckson DS. Estmaton of the concentraton of low-densty lpoproten cholesterol n plasma, wthout use of the preparatve ultracentrfuge. Cln Chem. 1972;18: DeLong DM, DeLong ER, Wood PD, Lppel K, Rfknd BM. A comparson of methods for the estmaton of plasma low- and very low-densty lpoproten cholesterol: The Lpd Research Clncs Prevalence Study. JAMA. 1986;256: McNamara JR, Cohn JS, Wlson PWF, Schaefer EJ. Calculated values for low densty lpoproten cholesterol n the assessment of lpd abnormaltes and coronary dsease rsk. Cln Chem. 199;36:36-^2. 8. McNamara R, Cole TG, Contos JH, et al. Immunoseparaton method for measurng low-densty lpoproten cholesterol drectly from serum evaluated. Cln Chem. 1995;41: Ransdell J, Slone T, Clfton D, Rchardson V, Hodges D, Oeltgen P. Clncal evaluaton of a drect LDL cholesterol mmunoseparaton kt. Cln Chem. 1994;4: Sngh J, Kulg K, Lucco L. Determnaton of drect low densty lpoproten cholesterol (LDL) n human serum wth trglycerdes <4 mg/dl by Genzyme mmunoseperaton reagent (Gen-LDL) kt. Cln Chem. 1994;4: Castellan WJ, Ghadal M. Evaluaton of the Sgma dagnostcs method for drect low-densty cholesterol measurement by mmunoprecptaton. Cln Chem. 1994;4: Schectman G, Sasse E. Varablty of lpd measurements: relevance for the clncan. Cln Chem. 1993;39: Hostetter AL. Screenng for dyslpdema: Practce parameter. Am J Cln Pathol. 1995;13: Cole T, Ferguson CA. Evaluaton of drect LDL cholesterol method. Atheroscleross. 1994;18:S191 (abstract). 15. Lpd Research Clncs Program: The Lpd Research Clncs Coronary Prmary Preventon Tral results: II. The relatonshp of reducton n ncdence of coronary heart dsease to cholesterol lowerng. JAMA. 1984;251: Unted States General Accountng Offce. Cholesterol measurement: Test accuracy and factors that nfluence cholesterol levels. Washngton, DC: Unted States General Accountng Offce; December, GAO/PEMD E 617