Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Metabolic syndrome (MetS) adults (n = 24; 18-65 y) will be enrolled to complete a 2-arm, double-blind, randomized controlled, crossover trial. They will be randomized in 4-unit blocks to receive, for 13 d, a controlled diet with dairy milk (3.5% fat; 3 servings/d) enriched with milk fat globule membrane (MFGM, MEB) or a matched dairy milk that instead contains soy lecithin/phospholipid (control, COMP). All foods during each study period will be provided to ensure weight maintenance and to increase homogeneity of gut and host responses. Anthropometrics and blood pressure will be assessed at days 0, 7, and 13. Prior to (day 0) and after each 2-wk arm (day 13), a fasting blood sample will be collected to assess serum endotoxin and metabolic chemistries (glucose, lipids, insulin), and Toll-like receptor 4 /nuclear factor kappaB (TLR4/NFκB)-dependent genes from whole blood. A breath sample will be collected to assess the correlation analysis of plasma metabolic biomarkers. After the 2-week intervention, from fecal samples collected on day 13, the investigators will assess microbiota composition and function, short chain fatty acids (SCFA), and intestinal inflammatory markers (calprotectin, myeloperoxidase). On d 13, participants in the fasted state will receive a high-fat/high-glucose meal challenge to induce gut-derived endotoxin translocation. At 30-minute intervals for 3-hour, the investigators will evaluate circulating endotoxin, glucose, and insulin; TLR4/NFκB-dependent genes will be assessed from whole blood at 0 hour and 3-hour. Gut permeability probes will be co-administered with the test meal challenge, and 24-hour urine will be collected to assess gut barrier integrity. Participants will then undergo a 2-week washout prior to receiving the alternative treatment and completing all procedures in an identical manner.
Background and hypothesis:
Our preclinical evidence shows that phospholipid-rich milk fat globule membrane (MFGM) attenuates lipopolysaccharide-induced increases in gut permeability and pro-inflammatory cytokines. MFGM also attenuates inflammation in association with a prebiotic and/or antimicrobial activity that modulates microbiota composition. Our central hypothesis is that MFGM-enriched dairy milk compared with a matched milk beverage containing soy lecithin (control) decreases metabolic endotoxemia and improves glucose tolerance in metabolic syndrome (MetS) adults by increasing gut barrier integrity in association with alleviating gut dysbiosis and inflammation.
Study Design:
The investigators will enroll male and female MetS adults (n = 24; 18-65 y) to complete a 2-arm, double-blind, randomized controlled, crossover trial. They will be randomized in 4-unit blocks to receive, for 14 days, a controlled diet with dairy milk (3.5% fat; 3 servings/d) enriched with MFGM-derived phospholipid or a matched dairy milk that instead contains coconut and palm oil (control). The investigators will provide all foods during each study period to ensure weight maintenance and to increase homogeneity of gut and host responses.
Subjects:
Participants will be recruited from Columbus, Ohio area. Participants having no history of liver or cardiovascular disease or cancer will be enrolled. They will have ≥3 established criteria for MetS: i) glucose (100-126 mg/dL), ii) waist circumference (>89 or >102 cm for F/M), iii) HDL-C (<50 or <40 mg/dL in F/M), iv) triglyceride >150 mg/dL, and iv) blood pressure >130/85 mmHg. Major exclusion criteria include: unstable body mass (±2 kg over prior 3-mo) vegetarian; food allergies or lactose intolerance; user of dietary supplements or probiotics (within past 1-mo); pregnancy, lactation, changes in birth control (within 6-month); any gastrointestinal disorders; chronic diarrhea; smoker; excess alcohol (>2 drinks/day); excess aerobic exercise (>7 h/week); recent antibiotic or anti-inflammatory agent use; blood pressure >140/90 mmHg.
Dietary Control:
The intervention will be performed in the Human Nutrition Metabolic Kitchen under the auspice of a registered dietitian (PI Bruno). In each 2-wk intervention, participants' diet will be rigorously controlled. All foods will be prepared, packaged, and provided every 3-4 days to supply a weight maintenance (i.e. eucaloric) diet. To assess compliance, participants will return MFGM/coconut/palm oil milk bottles for counting and any uneaten food portions for weighed measurement. Milk beverages will also be formulated to contain para-aminobenzoic acid (PABA; 80 mg/milk serving). Spot urine samples will be collected 4 times during each study arm coinciding when participants pick up test foods. Urinary PABA will be measured by spectrophotometry. Separate from this, participants will also keep food logs to document any dietary deviation. Diets will be standardized at 50-60% of energy from carbohydrate with low fiber intakes (~15 g/day) similar to Americans' diets to prevent potential masking of the benefits of MFGM, 15-20% from protein, and 25-30% from fat. Importantly, other than test beverages provided as part of the eucaloric diet, diets will be otherwise devoid of significant amounts of dairy foods, fermented products, and probiotics to prevent confounding effects.
Measurements:
Anthropometrics and blood pressure will be assessed at days 0, 7, and 13. Prior to (day 0), at day 7 and after each 2-wk arm (day 13), a fasting blood sample will be collected to assess serum endotoxin and metabolic chemistries (glucose, insulin, lipids (triglyceride, total and HDL cholesterol), and TLR4/NFκB-dependent genes from whole blood. A breath sample will be collected to assess the correlation analysis of plasma metabolic biomarkers. After the 2-week intervention, from fecal samples collected on day 13, the investigators will assess microbiota composition and function, SCFAs, and intestinal inflammatory markers (calprotectin, myeloperoxidase). During this period, participants will also record daily stool characteristics using a 7-point Bristol Stool scale. On day 13, participants in the fasted state will receive a high-fat/high-glucose meal challenge to induce gut-derived endotoxin translocation. At 30-minute intervals for 3 hours, the investigators will evaluate circulating endotoxin, glucose, and insulin; TLR4/NFκB-dependent genes will be assessed from whole blood at 0 hour and 3-hour. Gut permeability probes will be co-administered with the test meal challenge, and 24-hour urine will be collected to assess gut barrier integrity. Participants will then undergo a 2-week washout prior to receiving the alternative treatment and completing all procedures in an identical manner.
Not provided
Not provided
Not provided
Not provided
| Label | Type | Description | Intervention Names |
|---|---|---|---|
| MFGM-enriched full-fat dairy milk | Experimental | Participants in this arm will receive MFGM-enriched full-fat dairy milk (3 servings/d) that contains MFGM at 10% phospholipid (relative to total lipid content) delivering MFGM at ~10-times that in full-fat dairy milk. |
|
| Soy phospholipid/lecithin milk | Placebo Comparator | Participants in this arm will receive a matched dairy milk that instead contains soy phospholipid/lecithin. |
|
| Name | Type | Description | Arm Group Labels | Other Names |
|---|---|---|---|---|
| MFGM-enriched full-fat dairy milk | Dietary Supplement | MetS adults with metabolic endotoxemia will be enrolled to complete a 2-arm, double-blind, randomized controlled, crossover trial to test the independent benefits of MFGM. For each 2-wk arm, they will receive MFGM-enriched full-fat dairy milk (3 servings/d) or a matched dairy milk that instead contains soy phospholipid/lecithin (control). Milks have been formulated with soy lecithin or MFGM at 10% phospholipid (relative to total lipid content). This delivers MFGM at ~10-times that in full-fat dairy milk, which reflects that consumers obtain MFGM from dairy foods other than whole milk. |
| Measure | Description | Time Frame |
|---|---|---|
| Serum Endotoxin | Data are biomarker fasting concentrations | Day 13 |
| Serum Endotoxin | Data are biomarker area under the time-concentration curve (0-3 hours) on day 13 | Day 13 (0, 30, 60, 90, 120, 150, 180 minutes post-meal challenge) |
| Measure | Description | Time Frame |
|---|---|---|
| Plasma Glucose | Data are biomarker area under the time-concentration curve (0-3 hours) on day 13 | Day 13 (0, 30, 60, 90, 120, 150, 180 minutes post-meal challenge) |
| Plasma Glucose | Data are biomarker fasting concentrations |
Not provided
Inclusion Criteria:
Exclusion Criteria:
Not provided
Not provided
Not provided
Not provided
Not provided
| Name | Affiliation | Role |
|---|---|---|
| Richard Bruno, PhD | Ohio State University | Principal Investigator |
| Facility | Status | City | State | ZIP | Country | Contacts |
|---|---|---|---|---|---|---|
| The Ohio State University | Columbus | Ohio | 43201 | United States |
| PubMed Identifier | Type | Citation | Retractions |
|---|---|---|---|
| 38669850 | Background | Pokala A, Kraft J, Taormina VM, Michalski MC, Vors C, Torres-Gonzalez M, Bruno RS. Whole milk dairy foods and cardiometabolic health: dairy fat and beyond. Nutr Res. 2024 Jun;126:99-122. doi: 10.1016/j.nutres.2024.03.010. Epub 2024 Mar 21. | |
| 34879146 | Background | Bruno RS, Pokala A, Torres-Gonzalez M, Blesso CN. Cardiometabolic health benefits of dairy-milk polar lipids. Nutr Rev. 2021 Dec 8;79(Suppl 2):16-35. doi: 10.1093/nutrit/nuab085. |
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
Not provided
| ID | Title | Description |
|---|---|---|
| FG000 | MFGM Enriched Full-fat Dairy Milk Then Soy Phospholipid/Lecithin Milk | Participants in this sequence first received Milk fat globule membrane (MFGM) enriched full-fat dairy milk (3 servings/day for 2 weeks), followed by a 2-week washout, and then soy phospholipid/lecithin milk (3 servings/day for 2 weeks). |
| FG001 | Soy Phospholipid/Lecithin Milk Then MFGM-enriched Full-fat Dairy Milk | Participants in this sequence first received soy phospholipid/lecithin milk (3 servings/day for 2 weeks), followed by a 2-week washout, and then MFGM-enriched full-fat dairy milk (3 servings/day for 2 weeks). |
| Title | Milestones | Reasons Not Completed | |||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| First Intervention (2 Weeks) |
|
| |||||||||||||||||||||
| Washout (2 Weeks) |
| ||||||||||||||||||||||
| Second Intervention (2 Weeks) |
|
Not provided
Not provided
| ID | Title | Description |
|---|---|---|
| BG000 | All Study Participants | Baseline characteristics of Metabolic Syndrome (MetS) participants at screening |
| Units | Counts |
|---|---|
| Participants |
|
| Title | Description | Population Description | Parameter Type | Dispersion Type | Unit of Measure | Calculate Percentage | Denominator Units Selected | Denominators | Classes |
|---|---|---|---|---|---|---|---|---|---|
| Age, Categorical | Count of Participants |
| Type | Title | Description | Population Description | Reporting Status | Anticipated Posting Date | Parameter Type | Dispersion Type | Unit of Measure | Calculate Percentage | Time Frame | Units Analyzed | Denominator Units Selected | Arm/Group Information | Denominators | Classes | Analyses | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Primary | Serum Endotoxin | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | EU/ml | Day 13 |
|
3-months
Not provided
Not provided
| ID | Title | Description | Deaths (Affected) | Deaths (At Risk) | Serious Events (Affected) | Serious Events (At Risk) | Other Events (Affected) | Other Events (At Risk) |
|---|---|---|---|---|---|---|---|---|
| EG000 | MFGM-enriched Full-fat Dairy Milk | MetS adults with metabolic endotoxemia will be enrolled to complete a 2-arm, double-blind, randomized controlled, crossover trial to test the independent benefits of MFGM. For each 2-wk arm, they will receive either MFGM-enriched full-fat dairy milk (3 servings/d) or a matched dairy milk containing soy phospholipids/lecithin (control). Milks have been formulated with soy lecithin or MFGM at 10% phospholipid (relative to total lipid content). |
Not provided
Not provided
Not provided
| Title | Organization | Phone | Extension | |
|---|---|---|---|---|
| Richard Bruno | Ohio State University | 8603358494 | bruno.27@osu.edu |
Not provided
| Type | Includes Protocol | Includes SAP | Includes ICF | Document Label | Document Date | Document Uploaded Date | Document File Name |
|---|---|---|---|---|---|---|---|
| Prot_SAP | Yes | Yes | No | Study Protocol and Statistical Analysis Plan | Oct 18, 2018 | Jul 13, 2025 | Prot_SAP_000.pdf |
Not provided
| ID | Term |
|---|---|
| D024821 | Metabolic Syndrome |
| D007249 | Inflammation |
| D009765 | Obesity |
| ID | Term |
|---|---|
| D007333 | Insulin Resistance |
| D006946 | Hyperinsulinism |
| D044882 | Glucose Metabolism Disorders |
| D008659 | Metabolic Diseases |
Not provided
Not provided
2-arm, double-blind, randomized controlled, crossover trial
Not provided
Not provided
Both participants and key research personnel will be blinded between MFGM-enriched full-fat dairy milk (treatment) and milk with soy phospholipid/lecithin (control).
|
| Soy phospholipid/lecithin milk | Dietary Supplement | MetS adults with metabolic endotoxemia will be enrolled to complete a 2-arm, double-blind, randomized controlled, crossover trial to test the independent benefits of MFGM. For each 2-wk arm, they will receive MFGM-enriched full-fat dairy milk (3 servings/d) or a matched dairy milk that instead contains soy phospholipid/lecithin (control). Milks have been formulated with soy lecithin or MFGM at 10% phospholipid (relative to total lipid content). |
|
| Day 13 |
| Plasma Insulin | Data are biomarker fasting concentrations | Day 13 |
| Plasma Insulin | Data are biomarker area under the time-concentration curve (0-3 hours) on day 13 | day 13 (0, 30, 60, 90, 120, 150, 180 minutes post-meal challenge) |
| Plasma HDL-C | Data are biomarker fasting concentrations | Day 13 |
| Plasma Triglyceride | Data are biomarker fasting concentrations | Day 13 |
| Plasma Total Cholesterol | Data are biomarker fasting concentrations | Day 13 |
| Plasma Glucagon-like Peptide-1 | Data are biomarker fasting concentrations | Day 13 |
| Urine Lactulose/Mannitol | Data are the 24-hour urinary excretion ratio of lactulose relative to mannitol post-meal challenge on day 13 | Day 13 |
| Urine Sucralose/Erythritol | Data are the 24-hour urinary excretion ratio of sucralose relative to erythritol post-meal challenge on day 13 | 5-24 hours |
| Fecal Acetate | Data are biomarker fecal concentrations | Day 13 |
| Fecal Butyrate | Data are biomarker fecal concentrations | Day 13 |
| Fecal Propionate | Data are biomarker fecal concentrations | Day 13 |
| Plasma Gastric Inhibitory Polypeptide | Data are biomarker fasting concentrations | Day 13 |
| Fecal Calprotectin | Data are biomarker fecal concentrations | Day 13 |
| Fecal Myeloperoxidase | Data are biomarker fecal concentrations | Day 13 |
| Whole-blood Messenger RNA Expression of Toll-like Receptor-4 at Fasting | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels. | Day 13 |
| Whole-blood Messenger RNA Expression of Interleukin-6 at Fasting | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels. | Day 13 |
| Whole Blood Messenger RNA Expression of Tumor Necrosis Factor-alpha at Fasting | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels. | Day 13 |
| Whole Blood Messenger RNA Expression of Toll-like Receptor-4 at 3 h Post-meal Challenge | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels measure at 3-hours post-meal challenge | Day 13, 3 h post-meal |
| Whole Blood Messenger RNA Expression of Interleukin-6 at 3 h Post-meal Challenge | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels measure at 3-hours post-meal challenge | Day 13, 3 h post-meal |
| Whole Blood Messenger RNA Expression of Tumor Necrosis Factor-alpha at 3 h Post-meal Challenge | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels measure at 3-hours post-meal challenge | Day 13, 3 h post-meal |
| 32885133 | Background | Quarles WR, Pokala A, Shaw EL, Ortega-Anaya J, Hillmann L, Jimenez-Flores R, Bruno RS. Alleviation of Metabolic Endotoxemia by Milk Fat Globule Membrane: Rationale, Design, and Methods of a Double-Blind, Randomized, Controlled, Crossover Dietary Intervention in Adults with Metabolic Syndrome. Curr Dev Nutr. 2020 Jul 25;4(9):nzaa130. doi: 10.1093/cdn/nzaa130. eCollection 2020 Sep. |
| 37513677 | Result | Pokala A, Quarles WR, Ortega-Anaya J, Jimenez-Flores R, Cao S, Zeng M, Hodges JK, Bruno RS. Milk-Fat-Globule-Membrane-Enriched Dairy Milk Compared with a Soy-Lecithin-Enriched Beverage Did Not Adversely Affect Endotoxemia or Biomarkers of Gut Barrier Function and Cardiometabolic Risk in Adults with Metabolic Syndrome: A Randomized Controlled Crossover Trial. Nutrients. 2023 Jul 23;15(14):3259. doi: 10.3390/nu15143259. |
| NOT COMPLETED |
|
| NOT COMPLETED |
|
|
| Participants |
|
| Sex: Female, Male | Count of Participants | Participants |
|
| Race and Ethnicity Not Collected | Race and Ethnicity were not collected from any participant. | Count of Participants | Participants |
|
| Region of Enrollment | Number | participants |
|
| Body Mass Index (kg/m2) | Mean | Standard Deviation | kg/m^2 |
|
| Waist circumference (cm) | Mean | Standard Deviation | cm |
|
| Systolic blood pressure (mmHg) | Mean | Standard Deviation | mmHg |
|
| Diastolic blood pressure (mmHg) | Mean | Standard Deviation | mmHg |
|
| Plasma Triglyceride (mg/dL) | Mean | Standard Deviation | mg/dL |
|
| Plasma HDL-C (mg/dL) | Mean | Standard Deviation | mg/dL |
|
| Plasma glucose (mg/dL) | Mean | Standard Deviation | mg/dL |
|
| Units | Counts |
|---|---|
| Participants |
|
|
| Primary | Serum Endotoxin | Data are biomarker area under the time-concentration curve (0-3 hours) on day 13 | Posted | Mean | Standard Error | EU/ml x min | Day 13 (0, 30, 60, 90, 120, 150, 180 minutes post-meal challenge) |
|
|
|
| Secondary | Plasma Glucose | Data are biomarker area under the time-concentration curve (0-3 hours) on day 13 | Posted | Mean | Standard Error | mg/dL x min | Day 13 (0, 30, 60, 90, 120, 150, 180 minutes post-meal challenge) |
|
|
|
| Secondary | Plasma Glucose | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | mg/dL | Day 13 |
|
|
|
| Secondary | Plasma Insulin | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | uIU/mL | Day 13 |
|
|
|
| Secondary | Plasma Insulin | Data are biomarker area under the time-concentration curve (0-3 hours) on day 13 | Posted | Mean | Standard Error | uIU/mL x min | day 13 (0, 30, 60, 90, 120, 150, 180 minutes post-meal challenge) |
|
|
|
| Secondary | Plasma HDL-C | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | mg/dL | Day 13 |
|
|
|
| Secondary | Plasma Triglyceride | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | mg/dL | Day 13 |
|
|
|
| Secondary | Plasma Total Cholesterol | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | mg/dL | Day 13 |
|
|
|
| Secondary | Plasma Glucagon-like Peptide-1 | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | pmol/L | Day 13 |
|
|
|
| Secondary | Urine Lactulose/Mannitol | Data are the 24-hour urinary excretion ratio of lactulose relative to mannitol post-meal challenge on day 13 | Posted | Mean | Standard Error | ratio | Day 13 |
|
|
|
| Secondary | Urine Sucralose/Erythritol | Data are the 24-hour urinary excretion ratio of sucralose relative to erythritol post-meal challenge on day 13 | Posted | Mean | Standard Error | ratio | 5-24 hours |
|
|
|
| Secondary | Fecal Acetate | Data are biomarker fecal concentrations | Posted | Mean | Standard Error | umol/g | Day 13 |
|
|
|
| Secondary | Fecal Butyrate | Data are biomarker fecal concentrations | Posted | Mean | Standard Error | umol/g | Day 13 |
|
|
|
| Secondary | Fecal Propionate | Data are biomarker fecal concentrations | Posted | Mean | Standard Error | umol/g | Day 13 |
|
|
|
| Secondary | Plasma Gastric Inhibitory Polypeptide | Data are biomarker fasting concentrations | Posted | Mean | Standard Error | pg/mL | Day 13 |
|
|
|
| Secondary | Fecal Calprotectin | Data are biomarker fecal concentrations | Posted | Mean | Standard Error | ug/g | Day 13 |
|
|
|
| Secondary | Fecal Myeloperoxidase | Data are biomarker fecal concentrations | Posted | Mean | Standard Error | ng/g | Day 13 |
|
|
|
| Secondary | Whole-blood Messenger RNA Expression of Toll-like Receptor-4 at Fasting | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels. | Posted | Mean | Standard Error | log2(fold change) | Day 13 |
|
|
|
| Secondary | Whole-blood Messenger RNA Expression of Interleukin-6 at Fasting | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels. | Posted | Mean | Standard Error | log2(fold change) | Day 13 |
|
|
|
| Secondary | Whole Blood Messenger RNA Expression of Tumor Necrosis Factor-alpha at Fasting | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels. | Posted | Mean | Standard Error | log2(fold change) | Day 13 |
|
|
|
| Secondary | Whole Blood Messenger RNA Expression of Toll-like Receptor-4 at 3 h Post-meal Challenge | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels measure at 3-hours post-meal challenge | Posted | Mean | Standard Error | log2(fold change) | Day 13, 3 h post-meal |
|
|
|
| Secondary | Whole Blood Messenger RNA Expression of Interleukin-6 at 3 h Post-meal Challenge | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels measure at 3-hours post-meal challenge | Posted | Mean | Standard Error | log2(fold change) | Day 13, 3 h post-meal |
|
|
|
| Secondary | Whole Blood Messenger RNA Expression of Tumor Necrosis Factor-alpha at 3 h Post-meal Challenge | Data are expression levels of whole blood target gene normalized to housekeeping gene (β-actin) expression levels measure at 3-hours post-meal challenge | Posted | Mean | Standard Error | log2(fold change) | Day 13, 3 h post-meal |
|
|
|
| 0 |
| 30 |
| 0 |
| 30 |
| 0 |
| 30 |
| EG001 | Soy Phospholipid/Lecithin Milk | MetS adults with metabolic endotoxemia will be enrolled to complete a 2-arm, double-blind, randomized controlled, crossover trial to test the independent benefits of MFGM. For each 2-wk arm, they will receive either MFGM-enriched full-fat dairy milk (3 servings/d) or a matched dairy milk containing soy phospholipids/lecithin (control). | 0 | 30 | 0 | 30 | 0 | 30 |
Not provided
Not provided
| D009750 |
| Nutritional and Metabolic Diseases |
| D010335 | Pathologic Processes |
| D013568 | Pathological Conditions, Signs and Symptoms |
| D050177 | Overweight |
| D044343 | Overnutrition |
| D009748 | Nutrition Disorders |
| D001835 | Body Weight |
| D012816 | Signs and Symptoms |