# US 11,442,072 B2 · Method for measuring LDL aggregation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,442,072 B2 - Document ID: US11442072B2 - Status: Granted - Kind: B2 - Application number: 16/459,524 - Filed: 2019-07-01 - Published: 2020-01-23 - Granted: 2022-09-13 - Earliest claimed: 2018-07-10 - Assignees: Jenny and Antti Wihuri Foundation - Applicants: Jenny and Antti Wihuri Foundation - Inventors: Katariina Öörni; Maija Ruuth; Petri Kovanen - Web page: https://patentgrep.com/patent/US11442072B2 ## CPC - G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} - A23L 33/10: using additives (addition of substantially indigestible substances A23L33/21) - A23L 33/40: {Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula} - A61K 31/575: substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol - C12Q 1/44: involving esterase - C12Q 1/60: involving cholesterol - A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present disclosure relates to a method for measuring aggregation of low-density lipoprotein (LDL). The susceptibility of LDL to aggregate is an important factor regarding prognosis, diagnosis and surveillance of atherosclerotic cardiovascular diseases and cardiometabolic diseases, such as type 2 diabetes and related conditions, at various stages of their development. Also provided are methods of treatment of such diseases in subjects in need thereof. ## Claims ### Claim 1 A method of determining a presence or risk of an atherosclerotic cardiovascular disease (ASCVD) or a cardiometabolic disease in a human subject, the method comprising: providing LDL particles from a blood plasma or serum sample of the human subject; mixing the LDL particles with a sphingomyelinase enzyme in a solution at acidic pH; detecting formation of LDL aggregates in the solution, and measuring the sizes of the detected LDL aggregates, wherein the detected LDL aggregates have a median size of at least 200 nm and the human subject is determined to have or be at risk for developing an ASCVD or a cardiometabolic disease. ### Claim 2 (depends on 1) The method of claim 1, wherein the step of providing LDL particles from a human blood plasma or serum sample comprises isolating LDL particles from the human plasma or serum sample by ultracentrifugation. ### Claim 3 (depends on 1) The method of claim 1, further comprising, prior to mixing, determining the size of the LDL particles. ### Claim 4 (depends on 1) The method of claim 1, wherein the acidic pH is a pH of between 5 and 6. ### Claim 5 (depends on 1) The method of claim 1, comprising mixing the LDL particles with the sphingomyelinase enzyme in a solution at acidic pH at a temperature of about 30-40° C. ### Claim 6 (depends on 1) The method of claim 1, comprising detecting formation of LDL aggregates in the solution using dynamic light scattering (DLS) or UV-vis absorbance spectroscopy. ### Claim 7 (depends on 1) The method of claim 1, comprising measuring the size of the detected LDL aggregates every 15 to 30 minutes for up to 6 hours after mixing the LDL particles with the sphingomyelinase enzyme. ### Claim 8 (depends on 1) The method of claim 1, comprising measuring the size of the detected LDL aggregates about 2 hours after mixing the LDL particles with the sphingomyelinase enzyme. ### Claim 9 (depends on 1) The method of claim 1, wherein the human subject is administered a treatment for ASCVD or cardiometabolic disease. ### Claim 10 (depends on 1) The method of claim 1, comprising administering to the human subject a food, diet, or supplement comprising a plant stanol or plant stanol ester. ### Claim 11 A method comprising: providing a human plasma or serum sample; mixing the human plasma or serum sample with a sphingomyelinase enzyme at acidic pH; detecting formation of LDL aggregates in the sample, and measuring the sizes of the detected LDL aggregates. ### Claim 12 (depends on 11) The method of claim 11, wherein the detected LDL aggregates have a median size of at least 200 nm. ### Claim 13 A method comprising: providing LDL particles from a blood plasma sample of a human subject that has been taking a cholesterol lowering medication selected from the group consisting of HMG CoA reductase inhibitor, a selective cholesterol absorption inhibitor, and a bile-acid binding resin; mixing the LDL particles with sphingomyelinase enzyme in a solution at acidic pH; detecting formation of LDL aggregates in said solution; measuring the sizes of the detected LDL aggregates; and if the LDL aggregates have a median size of at least 500 nm, continue administering to the human subject a therapeutically effective amount of the cholesterol lowering medication in combination with a healthy Nordic diet, a low sucrose diet, or a Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) inhibitor; and if the LDL aggregates have a median size of below 500 nm, continue administering to the human subject a therapeutically effective amount of the cholesterol lowering medication. ### Claim 14 (depends on 13) The method of claim 13, further comprising administering to the human subject regardless of the median size of LDL aggregates a food, diet, or supplement comprising a plant stanol or plant stanol ester. ### Claim 15 (depends on 14) The method of claim 14, wherein the plant stanol or plant stanol ester is administered to the human subject at an amount of 1 gram to 3 grams daily. ### Claim 16 (depends on 13) The method of claim 13, wherein the human subject has an ASCVD or a cardiometabolic disease. ### Claim 17 A method comprising: providing LDL particles from a blood plasma sample of a human subject that has been taking a cholesterol lowering medication selected from the group consisting of HMG CoA reductase inhibitor, a selective cholesterol absorption inhibitor, and a bile-acid binding resin; mixing the LDL particles with sphingomyelinase enzyme in a solution at acidic pH; detecting formation of LDL aggregates in said solution; measuring the sizes of the detected LDL aggregates; and if the LDL aggregates have a median size of at least 500 nm, continue administering to the human subject a therapeutically effective amount of the cholesterol lowering medication in combination with a healthy Nordic diet, a PCSK9 inhibitor, or a plant stanol or plant stanol ester; and if the LDL aggregates have a median size of below 500 nm, continue administering to the human subject a therapeutically effective amount of the cholesterol lowering medication. ### Claim 18 A method comprising: providing LDL particles from blood plasma or serum samples of (a) a human subject at risk for developing ASCVD or a cardiometabolic disease and (b) a control human subject who is known to not have ASCVD or a cardiometabolic disease; mixing the LDL particles with a sphingomyelinase enzyme in a solution at acidic pH; detecting formation of LDL aggregates in the solution; measuring the sizes of the detected LDL aggregates every 15 to 30 minutes for up to 6 hours after mixing the LDL particles with the sphingomyelinase enzyme, and comparing the sizes of the detected LDL aggregates of the human subject at risk for developing ASCVD or a cardiometabolic disease with the sizes of LDL aggregates of the control human subject.