The non-HDL-C to APOB ratio as a predictor of inaccurate LDL-C measurement in patients with chronic intrahepatic cholestasis and jaundice: a retrospective study

Patients

We collected data from the First Affiliated Hospital of Guangzhou University of Chinese Medicine, including patients admitted between January 2020 and January 2023, who were diagnosed with intrahepatic cholestasis. The patients were grouped according to specific inclusion criteria. The inclusion criteria were based on the 2009 guidelines for the diagnosis and management of cholestatic liver diseases published by European Association for the Study of the Liver (2009). These criteria included alkaline phosphatase (ALP) levels higher than 1.5 times the upper limit of normal (ULN) and gamma-glutamyl transferase (GGT) levels higher than three times the ULN. Additionally, cholestatic liver disease patients were further categorized into cholestatic jaundice group (total bilirubin, TB, level ≥ 51.3 µmol/L) and cholestatic non-jaundice group (TB level < 51.3 µmol/L). Our study excluded patients with renal insufficiency, defined as the presence of either acute kidney injury (AKI) or chronic kidney disease (CKD) as determined by clinical diagnosis and renal function tests. Additionally, we excluded pregnant or lactating women, individuals under 18 years of age, patients with incomplete data on prior biliary surgery, and those with triglyceride levels above 4.52 mmol/L or LDL-C levels below 1.81 mmol/L. To ensure our findings were not influenced by lipid-lowering treatments, we also excluded patients who had received such medications within the 6 months prior to the study.

A total of 652 intrahepatic cholestasis patients were included in the study, of which 118 were in the cholestatic jaundice group (63 males and 55 females) and 534 were in the cholestatic non-jaundice group (275 males and 259 females). The study was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou University of Chinese Medicine (K-2023-135) and was conducted in accordance with the Declaration of Helsinki. Depending on the nature of the research, the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine has waived the need for consent.

Data collection

Clinical data of hospitalized patients with cholestasis presenting for the first time were collected, including gender and age. Additionally, fasting venous blood samples of 3 mL were collected using Polyethylene Terephthalate vacuum procoagulant tubes. The collected blood samples were centrifuged at approximately 4,100 × g for 5 min using a medical centrifuge, and the resulting serum was used for laboratory testing of various parameters. All laboratory tests were performed using the ROCHE Cobas 8000 fully automated biochemical analyzer. The reagents used included the following kits provided by Roche Diagnostics (Shanghai, China) Co., Ltd.: Triglyceride Test Kit (GPO-PAP enzymatic colorimetric method), Cholesterol Test Kit (CHOD-PAP enzymatic colorimetric method), High-Density Lipoprotein Cholesterol Test Kit (selective inhibition method, PPD method), Low High-Density Lipoprotein Cholesterol Test Kit (solubilization method, SOL method), Gamma-Glutamyl Transferase Test Kit (γ-glutamyl-3-carboxy-4-nitroaniline rate method), and Alkaline Phosphatase Test Kit (AMP buffer rate method). The ApoA1/ApoB Test Kit (immunoturbidimetric method) was provided by North Control Bio-Tech Co., Ltd, (Sichuan, China). The Total Bilirubin Test Kit (vanadate method) and Direct Bilirubin Test Kit (vanadate method) were provided by Maccura Biological Technology (Sichuan, China) Co., Ltd.

Calculation of LDL-C(F)

The LDL-C measurement obtained through instrument analysis is referred to as LDL-C (D). LDL-C(F) is calculated using the Friedewald equation based on the instrument-measured values of TC, TG, and HDL-C. The Friedewald equation is as follows: LDL-C(F) = TC–HDL-C–TG/2.2. If the percentage difference ((LDL-C(D)–LDL-C(F))/LDL-C(D) is equal to or greater than 30%, we consider the results of LDL-C(D) and LDL-C(F) to be inaccurate. Conversely, if the percentage difference is less than 30%, we consider the results to be accurate.

Calculation of NH-C/B ratio

The non-HDL-C to APOB ratio (NH-C/B ratio) is calculated using the formula $\frac{TC-HDL-C}{APOB}$, where TC denotes total cholesterol, HDL-C denotes high-density lipoprotein cholesterol, and APOB represents apolipoprotein B.

Statistical analysis

Statistical analysis was performed using SPSS 25.0 software (SPSS, Armonk, NY, USA). For continuous variables, data were expressed as mean ± standard deviation if they followed a normal distribution, and as median (interquartile range) (M(Q1-Q3)) if they did not. When comparing two groups, if the data followed a normal distribution, the unpaired t-test was used for statistical analysis. If the data did not follow a normal distribution, a nonparametric method was employed. A two-tailed P-value of less than 0.05 was considered statistically significant. Linear correlation analyses were conducted between LDL-C(D) and LDL-C(F), HDL-C and ApoA1, LDL-C(D) and ApoB, and LDL-C(F) and ApoB in both the cholestatic jaundice and cholestatic non-jaundice groups. If the data followed a normal distribution, Pearson correlation analysis was used. If the data did not follow a normal distribution, Spearman correlation analysis was utilized.

Intrahepatic cholestasis jaundice impacts LDL-C discrepancy

As shown in Fig. 1, a total of 652 cases of intrahepatic cholestasis were included in our study, with 118 cases in the cholestatic jaundice group and the remaining 534 cases in the cholestatic non-jaundice group. Furthermore, patients were categorized into acute and chronic groups based on whether the duration of cholestasis exceeded 6 months. Among the 118 cases with jaundice, there were 61 chronic and 57 acute cases; among the 534 cases without jaundice, there were 265 chronic and 269 acute cases. Laboratory test results for liver function and blood lipid parameters were collected for both groups. As shown in Table 1, there were significant differences in the laboratory test results of liver function and blood lipids between the two groups. Compared to the cholestatic non-jaundice group, the cholestatic jaundice group had higher levels of TC and TG, and lower levels of HDL-C. Interestingly, the proportion of patients in the cholestatic jaundice group with a discrepancy between LDL-C(F) and LDL-C(D) reached 36.4%, which was significantly higher than that in the cholestatic non-jaundice group.

Weak LDL-C correlations in intrahepatic cholestasis jaundice

Furthermore, we conducted a correlation analysis on certain lipid profile parameters. As shown in Fig. 2A, we found a strong correlation between HDL-C and ApoA1 in both the cholestatic non-jaundice and cholestatic jaundice groups. However, as depicted in Fig. 2B, the correlation coefficient between LDL-C(F) and LDL-C(D) in the cholestatic jaundice group was 0.633, indicating a relatively weak correlation, whereas in the cholestatic non-jaundice group, these two variables exhibited a strong correlation. Further analysis revealed, as shown in Figs. 2C and 2D, a relatively weak correlation between LDL-C(F) and ApoB, as well as between LDL-C(D) and ApoB in the cholestatic jaundice group, with correlation coefficients significantly lower than those observed in the cholestatic non-jaundice group.

NH-C/B ratio and TC as predictors for LDL-C accuracy

In order to identify additional predictors of inaccurate LDL-C measurements in patients with intrahepatic cholestatic jaundice, we conducted further analysis of laboratory data collected from 118 cases in the cholestatic jaundice group, as presented in Table 2. Univariate and multivariate logistic regression analyses revealed that TC, TG, ALP and NH-C/B ratio were independent markers for predicting inaccurate LDL-C measurements in patients with cholestatic jaundice.

Furthermore, we conducted ROC curve analysis to evaluate the diagnostic performance of TC, TG, ALP and NH-C/B ratio as predictors of inaccurate LDL-C measurements in patients with intrahepatic cholestatic jaundice. As shown in Fig. 3A, the results indicated that TC and NH-C/B ratio demonstrated relatively good diagnostic efficacy, with areas under the curve (AUCs) of 0.770. To further investigate the diagnostic performance of these two parameters in acute and chronic intrahepatic cholestasis, we conducted separate analyses. The results, as shown in Fig. 3B, revealed that in chronic intrahepatic cholestasis, the NH-C/B ratio showed relatively better diagnostic efficacy compared to TC, with an AUC of 0.770 (95% CI [0.641–0.900]). Utilizing a cut-off value of 4.50 for the NH-C/B ratio, the sensitivity was 62.5% and the specificity was 83.8%. On the other hand, as depicted in Fig. 3C, in acute intrahepatic cholestasis, TC exhibited relatively better diagnostic efficacy compared to the NH-C/B ratio, with an AUC of 0.818 (95% CI [0.709–0.927]). With a cut-off value of 5.03 mmol/L for TC, the sensitivity was 94.7% and the specificity was 68.4%.