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Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin–cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

Summary: Background: For many cardiovascular risk factors there is no lower limit to which further reduction will result in decreased disease risk; this includes values within ranges considered normal for healthy adults. This seems to be true for new emerging metabolic risk factors identified by innovative technological advances. Further, there seems to be ever evolving evidence of differential responses to lifestyle interventions by sex and body compositions in the normal range. In this secondary analysis, we had the opportunity to test these principles for newly identified molecular biomarkers of cardiometabolic risk in a young (21–50 years), normal weight healthy population undergoing calorie restriction for two years. Methods: The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) was a 24-month, multicenter, randomized controlled trial (May 2007-November 2012) in healthy, adults without obesity to evaluate the potential for calorie restriction (CR) to promote anti-aging adaptations, including those associated with disease risk. 218 participants (age 37.9 ± 7.2 years and body mass index (BMI) 25.1 ± 1.7 kg/m2, mean±SD) were randomized 2:1 to 24 months of CR (prescribed as 25% reduction from baseline calorie intake) versus ad libitum (AL). Fasting plasma from baseline, 12, and 24 months was used for assessments of lipoproteins, metabolites, and inflammatory markers using nuclear magnetic resonance spectroscopy. Findings: Averaging 11.9% CR, the CR group had reductions at 12 and 24 months in the cardiovascular disease risk markers, apolipoprotein B and GlycA, and risks for insulin resistance and type 2 diabetes—Lipoprotein Insulin Resistance Index and Diabetes Risk Index (all PCRvsAL≤0.0009). Insulin resistance and diabetes risk improvements resulted from CR-induced alterations in lipoproteins, specifically reductions in triglyceride-rich lipoprotein particles and low-density lipoprotein particles, a shift to larger high-density lipoprotein particles (more effective cholesterol transporters), and reductions in branched chain amino acids (BCAAs) (all PCRvsAL≤0.004). These CR responses were more pronounced in overweight than normal weight participants and greater in men than women. Interpretation: In normal to slightly overweight adults without overt risk factors or disease, 12 months of ∼12% CR improved newly identified risk markers for atherosclerotic cardiovascular disease, insulin resistance and type 2 diabetes. These markers suggest that CR improves risks by reducing inflammation and BCAAs and shifting lipoproteins from atherogenic to cholesterol transporting. Additionally, these improvements are greater for men and for those with greater BMIs indicating sex and BMI-influences merit attention in future investigations of lifestyle-mediated improvements in disease risk factors. Funding: The CALERIE™ trial design and implementation were supported by a National Institutes of Health (NIH) U-grant provided to four institutions, the three intervention sites and a coordinating center (U01 AG022132, U01 AG020478, U01 AG020487 U01 AG020480). For this secondary analysis including sample acquisition and processing, data analysis and interpretation, additional funding was provided by the NIH to authors as follows: R01 AG054840 (MO, VBK); R33 AG070455 (KMH, DCP, MB, SBR, CKM, LMR, SKD, CFP, CJR, WEK); P30 DK072476 (CKM, LMR); and U54 GM104940 (CKM, LMR).