RESUMO
Objective:A high performance liquid chromatography (HPLC) method was developed to determine the enzymatic activity of CD38 in blood, which was the major enzyme responsible for consuming nicotinamide adenine dinucleotide (NAD). Additionally, the study aimed to detect the differences in CD38 enzymatic activity among individuals of varying ages and health statuses.Methods:A 50 μl whole blood matrix and enzyme reaction substrate of 150 μl β-NAD at a concentration of 500 μmol/L were selected for the analysis. To eliminate the impact of endogenous β-NAD, the whole blood sample was pre-incubated at 37 ℃ for 20 minutes before adding the substrate. The reaction was terminated by perchloric acid (PCA) after incubation at 37 ℃ for 40 min. The change in product nicotinamide (NAM) before and after the enzymatic reaction was measured by HPLC to calculate the CD38 activity. The linearity, limit of detection, limit of quantification, precision, and stability of the method were evaluated. The CD38 enzymatic activities in 60 healthy volunteers and 30 colorectal cancer patients in blood were determined by the developed method.Results:Pre-incubation at 37 ℃ for 20 minutes eliminated the effect of endogenous β-NAD. The correlation coefficient of NAM was 0.999 in the concentration range of 0.1-3.2 μmol/L, with limit of detection of 0.5 nmol/L and limit of quantification of 2.1 nmol/L. The average within-run imprecision ( CV) and total CV were 3.22%-4.03% and 2.91%-4.70%, respectively. The recovery rate ranged from 94.82% to 96.81%. The CD38 activity of whole blood was stable by storage at 4 ℃ for 48 hours, storage at room temperature for 8 hours, thawing of frozen whole blood at room temperature for 2 hours, or repeated freeze-thawing three times. NAM, NAD standards, and pre-treatment samples were stable after 48 hours at 4 ℃ and 8 hours at room temperature. CD38 activity gradually decreased with increasing concentration of the added CD38 inhibitor 4-aminoquinoline derivative (78c). Measurement of 60 healthy physical examination population samples showed significantly higher CD38 enzyme activity in the elderly group than that in the young group ( t=-2.776, P=0.007) and measurement of 30 colorectal cancer patients showed significantly higher CD38 enzyme activity than that in healthy people ( t=-2.572, P=0.012). Conclusion:The established HPLC method for determining CD38 enzymatic activity is characterized by its simplicity, efficiency, accuracy, and reproducibility. This technique serves as a valuable tool for investigating aging and aging-related diseases.
RESUMO
Objective To propose and validate a reduced volume β-quantification method to measure serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Methods The reduced volume β-quantification method involved separation of LDL and HDL by ultracentrifugation and preparation of HDL by chemical precipitation. The sampling and reconstitution of the bottom fractions were performed gravimetrically and sample volume was thus decreased from 5 to 0.8 ml. High performance liquid chromatography was used to determine the cholesterol concentration of bottom fractions and HDL-C in the supernatant. Serum levels of LDL-C depended on a calculation of bottom fractions cholesterol minus HDL-C. Results The total CVs for HDL-C and LDL-C were 0.65％ -1.75％ and 0.63％ -1.11％. The results of the developed method were consistent with the current reference method and well within the allowable bias for Cholesterol Reference Method Laboratory Network surveys. Conclusion A new method for the measurement of HDL-C and LDL-C has been established. This method requires a small amount of serum and is easy to operate, exhibiting a desirable precision and accuracy. It is reliable and can be used as a candidate reference method for HDL-C and LDL-C.