Ronald P. Taylor,' Anthony V. Broccoli, and Charles M. Grisham University of Virginia Charlonesville. 22901
I I
Enzymatic and Colorimetric Determination of Total Serum Cholesterol An undergraduate biochemistry laboratory experiment
Atherosclerosis is characterized by the huild-up of cholesterol and cholesterol esters in the inner layers of large arteries (I). The accumulation of these lipids in major arteries can restrict the flow of blood to vital oreans and eventuallv lead to heart attack or stroke. It is therefore clear that clinical assavs of hlood cholesterol levels are of obvious imuortance in the, di:ignosis and tn:itttnent ot'the diseasr. h wrierv 01 inorranic rulorintetric assavs have bren developed over the years for the determinatibn of the level of cholesterol in the hlood (2). However, it has been noted that many of these techniques lack the overall specificity and ease of standardization which are of crucial importance in the oprr:uiun of a clinical laboratory assay. Recently Allain, et a1. I J Ih a w describtvl an elegnnt, simple enzymatic method for the detrrmin~tionof total serum cholesterol through the use of a singkt aqueuus reaEmr. The met hod is highly accurate and has l m n shown to he hiehl\ s ~ e c i i i cas wt,ll 134 ) and has ~s started to receive wide use in clinical assays. he importance of cholesterol in affectine the structural and dvnamic .moo. ertit:i of cell nteml~ranrsis also under acti\,e investigatinn in Intxwatories rhrouehout the world t5I and this assav has heen adopted for use invmany of these laboratories as well. In this communication we describe an undermaduate lahoratory experiment in which the student determines the level of cholesterol in serum both by a colorimetric experiment (the Lieherman-Burchard procedure) (2) and by following the procedures of Allain, e t al. (3).The latter method illustrates the ~ e n e r aprinciples l of a "coupled" enzyme assay and the use of thin layer chromatography for the qualitative identification of lipids. By comparing the results of the two independent determinations the student can evaluate the merits of each procedure. ~~~
~
..
~
Background Cholesterol is found in two forms in the blood: either as free c h ~ ~ l r s t e rodr. esterified w ~ t hlong chain fatty acids such as ~ ~ a l m i tacid. i c 'l'vt~~callv about 7 5 , of the serum cholesterol is esterified (6).These two forms of cholesterolare transported in the blood principally uia the lipoproteins, which have varying combinations of protein, choles&rol, cholesterol ester, and triglyceride (7). However, in the assay procedure the enzvmes involved (cholesterol ester hvdrolaie and cholesterol oxidase) are very specific and are able to act on the varying forms of cholesterol desuite the oresence of the nroteins and other constituents in the serum sample. The first step in the reaction involves the catalyzed hydrolysis of the cholesterol esters by cholesterol ester hydrolase to generate free cholesterol and the corresponding free fatty acid (see Figure 1, eqn. (1)). Free cholesterol is oxidized by cholesterol oxidase and molecular oxveen to give cholest-4en-%one and hydrogen peroxide (eqi. (2)). Finally, horseradish peroxidase couples the newly formed hydrogen peroxide to 4-aminoantipyrene and phenol to generate a highly colored quinoneimine dye which has its maximum absorbance a t 500 nm (c = 5.33.10Vmole of cholesterol). The reaction is run for exactly 10 min a t 37OC in 0.1 M phosphate buffer, p H 6.7. --
Hrwsnh Corwr Devul,,pmenr Awarderd the National l n s r ~ u w i d Health. Addrer: rorre;pmdcncr to thi5 authur.
q",",,"~,">"*
dVC
Reaction scheme for the enzymatic measurement of total serum cholesterol. CEH = holes sterol ester hydrolase: CO = cholesterol oxidase; HPOD = horseradish peroxidase.
Table 1. Ingredients for Serum Cholesterol Assay (3) Ingredient Sodium Cholate 4-Aminaantipyrine Phenol Na2HPOa NaH2POa Carbawax-6000 Cholesterol ester hydrolase Cholesterol oxidase Peroxidase # k c = 6.7
-
Concentration (mmoiell) 3 0.82 14 50 50 0.17 33 Ull 117Ull 67000 UII
+ 0.1.
Procedure An assay solution which contains all three enzymes and the other necessary reagents at the appropriate concentrationsshould be prepared a few hours in advance. Thesolution should be stable for about 24 hr if it is stored at 4 T o r for 8 hr if it isstored at room temperature. The orooer . , concentrationsof reaeents needed far a standardized assev ,we i-ttd in T,al,lr I , a d a nm[,letc kit h > r thr prrpnratwn of the . r . i ~ yr~.lut~c.n i a v a i l a h l e r ~ , m m c r c ~ e l l \ ~ ~ n t .kit T h,an r lwobtnintd in dn f ~ and ~ smh o d he st.tIde i u this t'
