We developed a completely automated fluorimetric method for the determination of cellular cholesterol, consisting of enzymatic hydrolysis of cholesteryl ester to free cholesterol and enzymatic oxidation of free cholesterol in the presence of an indicator substrate to produce a fluorescent product. For control preparations of monocytes, the mean detection limit was 2.57 μmol/5 × 105 cells and the mean within-batch coefficients of variation were 9.30, 600 and 3.73% at mean cholesterol concentrations of 1.94, 9.05 and 12.49 μmol/5 × 105 cells, respectively. The results correlated well with those obtained by gas-liquid chromatography.
HeiderJGBoyettRL. The picomole determination of free and total cholesterol in cells in culture. J Lipid Res1978; 19: 514–8.
2.
GambleWVaughanMKruthHSAviganJ.Procedure for determination of free and total cholesterol in micro-or nanogram amounts suitable for studies with cultured cells. J Lipid Res1978; 19: 1068–70.
3.
AkibaT.An enzymatic measurement of cholesterol with NAD-dependent cholesterol dehydrogenases and the reagents for the assay. Japanese patent1983; 19: 559–67.
4.
BoyumA.Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium. Scand J Immunol1983; 17: 429–36.
5.
BoyumA.Iodinated density gradient media. In: RickwoodD, ed. Practical Approaches in Biochemistry Series. Oxford: IRL Press, 1983: 147–71.
6.
ButtnerJBorthRBoutwellJHBroughtonPMGBowyerRC. Approved recommendation on quality control in clinical chemistry. Part 2. Assessment of analytical methods for routine use. Clin Chim Acta1979; 98: 145f–62f.
7.
FerrelWJRepolaDBatsakisJG. Variables in measurement of serum cholesterol by gas liquid chromatography. Clin Biochem1977; 10: 118–21.
8.
PollockMAJeffersonSGKaneJWLomaxKMacKinnonGWinnardCB. Method comparison—a different approach. Ann Clin Biochem1992; 29: 555–60.
9.
AllainCCPoonLSChanCSRichmondWFuPC. Enzymatic determination of total serum cholesterol. Clin Chem1974; 20: 470–5.
10.
HuangHKuanJWGuilbaultGG. Fluorometric enzymatic determination of total cholesterol in serum. Clin Chem1975; 21: 1605–8.
11.
LohmannWLohmannCH. Native fluorescence of platelets from patients with occlusive arterial disease. Biochem Biophys Res Commun1988; 152: 1410–5.
12.
McCombRBBondLWBurnettRWKeechRCBowersGN. Determination of the molar absorptivity of NADH. Clin Chem1976; 22: 141–50.
