METHODS OF PREPARING AND ANALYZING BREAST MILK

Abstract

The present disclosure provides methods of preparing breast milk for analysis analyzing breast milk, determining reference ranges for analytes in breast milk and testing breast milk for a panel of analytes. Aspects of the present disclosure provide dietary and lifestyle recommendations for the lactating mother-based on measured concentration of analytes. Retesting is disclosed to measure the influence of maternal diet on breast milk composition.

Claims

1. A method for analyzing breast milk for micronutrients comprising the steps of: a) collecting a human breast milk sample from a lactating individual; b) freezing the breast milk sample at a temperature between −20 degrees C. to −80 degrees C.; c) thawing the breast milk sample above 20 degrees C.; d) homogenizing the breast milk sample; e) combining the homogenized breast milk sample with hexane and sodium methoxide in methanol solution to form a mixture; f) vortexing the mixture to form a hexane layer; g) separating the hexane layer; h) filtering the hexane layer to produce a sample extract; and i) performing GC/MS analysis on the sample extract to provide measured micronutrient concentrations.

2. The method of claim 1 wherein the step of collecting a human breast milk sample comprises collection of breast milk over a 24 hour period to interrogate the entire feed of the lactating individual.

3. The method of claim 1 wherein the step of homogenizing the breast milk sample comprises sonicating the breast milk sample.

4. The method of claim 1 wherein step e) further comprises combining approximately one part mL amount of homogenized milk sample with approximately 20 parts hexane and two parts 5.4 M sodium methoxide in methanol solution.

5. The method of claim 4 wherein the measured micronutrient concentrations comprise one or more of Vitamin A, Vitamin D, Vitamin E, Vitamin K1, or Vitamin K2.

6. The method of claim further comprising the steps of: j) providing the lactating individual with the measured micronutrient concentrations; and k) comparing each measured micronutrient concentrations against a nutrient guidance concentration; and l) providing the lactating individual with dietary guidance for altering measured micronutrient concentrations.

7. The method of claim 6 further comprising retesting by repeating the steps a) through i) to determine the impact of dietary guidance on measured micronutrient concentrations.

8. A method for analyzing breast milk for fatty acid concentrations comprising the steps of: a) collecting a human breast milk sample from a lactating individual; b) freezing the breast milk sample at a temperature between −20 degrees C. to −80 degrees C.; c) thawing the breast milk sample above 20 degrees C.; d) homogenizing the breast milk sample; e) performing liquid liquid extraction by combining the breast milk sample with chloroform, methanol, and water in a ratio of approximately 1:2:0.8 in a separating vessel to form an upper layer and a lower layer; f) separating the lower layer containing chloroform and lipids; g) dissolving the lower layer in a nonpolar solvent; h) adding boron trichloride in methanol to form an extract mixture; i) heating the extract mixture at about 60 degrees C. for approximately ten minutes; j) cooling the extract mixture with water and hexane and missing; extracting an upper organic layer to provide a sample extract; and k) performing GC/MS analysis on the sample extract to provide measured fatty acid concentrations.

9. The method of claim 8 wherein the step of collecting a human breast milk sample comprises collection of breast milk over a 24 hour period to interrogate the entire feed of the lactating individual.

10. The method of claim 9 wherein the step of homogenizing the breast milk sample comprises sonicating the breast milk sample.

11. The method of claim 10 wherein the nonpolar solvent of step g) comprises hexane.

Description

DETAILED DESCRIPTION OF INVENTION

[0020] Reference will now be made in detail to the presently preferred embodiments of the invention. Analysis of human milk can be conducted with liquid chromatography with tandem mass spectrometry: LC/MS/MS, gas chromatography coupled with mass spectrometry (GC/MS), and/or analysis using inductively coupled plasma mass spectrometry (ICP-MS), or inductively coupled plasma with optical emission spectrometry (ICP-OES) analysis.

[0021] In some embodiments, the method of the invention includes collecting a milk sample (e.g., one or more samples) comprising breast milk from the human that produced the breast milk. In certain embodiments, collecting the sample includes extracting, from the human, an amount of breast milk into a test tube. Extracting breast milk from the human can be performed using known techniques, such as, but not limited to, commercially available breast milk pumps, pumping breast milk by hand, and the like. The sample collection container can comprise a sterile test tube. Specifically, 5-30 ml of human milk can be collected in a sterile test tube. A nursing individual can pool together all expressed samples throughout a 24-hour period.

[0022] The method can comprise refrigerating and then freezing the test tube containing the breast milk until the breast milk is ready to be analyzed. The step is necessary to preserve the chemical and physical properties of the macronutrients that need to be analyzed in the human milk (HM). The method can comprise sending the frozen breast milk with frozen gel packs to a laboratory for analyzing the breast milk. Dry ice is not needed for shipment. If the sample is not frozen adequately, endogenous lipases have the opportunity to cause lipid hydrolysis resulting in inaccurate and misrepresentative HM lipid content for measurement. Storage at −20 degrees C. or −80 degrees C. stops enzyme activity within the samples and HM lipid integrity is best preserved.

[0023] Upon receipt by a laboratory, a sample can be stored in a freezer from −20 to −80 degrees Celsius until ready for processing. Sample preparation differs depending on the analytical instrument that is used.

[0024] The method further provides for removing the sample from the freezer, thawing the sample at room temperature, for example at least 20 degrees C., and homogenizing to produce a prepared milk sample. The prepared milk sample is then ready for analysis.

Example 1

[0025] LC/MS analysis to measure micronutrients including Vitamin A, D, E, K1, K2, folate and choline, the sample requires a stepwise purification process. a) Combining approximately 0.5 mL amount of prepared milk sample with approximately 10 mL of hexane and 1 mL of 5.4 M sodium methoxide in methanol solution to form a mixture. b) Vortexing the sample to form a hexane layer in the test tube. c) Separating the hexane layer. d) Filtration of the hexane layer with a silica cartridge. The sample extract is ready for analysis.

Example 2

[0026] GC/MS Analysis is used to measure fatty acids. The initial step in the analysis of lipids present in HM is separation of lipids from the rest of HM components. Steps of the method comprise: a) Liquid-liquid extraction (LLE) is used to separate analytes by their relative solubility in different immiscible liquids. The first step consists in taking 1 mL of HM sample and to wash it with chloroform, methanol and water in ratio of 1:2:0.8 respectively in a separating funnel. The funnel is closed and shaken gently by inverting the funnel multiple times. The funnel is inverted and the stopcock carefully opened to release excess vapor pressure if present. The separating funnel is set aside to allow for the complete separation of the phases. The mixture (HM+chloroform, methanol and water) separates into the bottom the organic phase with chloroform and breast milk lipids, and into the top aqueous phase with methanol, water, and containing carbohydrates and salts. The top and the bottom stopcock are then opened and the lower phase is released by gravitation collecting the separated breast milk lipids dissolved in chloroform. Following the lipid extraction from the HM sample, the analyte undergoes a chemical transesterification. Hydrolysis of the triacylglycerides (TAGs) following the derivatization of the released fatty acids (FA) to methylesters (FAMEs) for GC analysis. The derivatization is necessary otherwise the GC column will give a false reading that will show a band broadening with a false retention time. b) Dissolve the analyte in a nonpolar solvent such as hexane in a vessel. c) Add 2 mL BCl3-methanol, 12% w/w. d) Heat at 60° C. for 10 minutes. e) Cool the sample, then add 1 mL water and 1 mL hexane and shake the vessel. f) After the layers settle, collect the upper organic layer to a clean vial to provide a sample extract. The sample extract is ready for injection into GC apparatus.

[0027] Calibration of GC can be conducted before analysis of the sample. Inject 1.0 μL FAMEs standard solution into GC apparatus. Observe chromatogram for any interferences and make sure that all FAMEs in the standard solution have eluted and that the 19 FAME peaks are resolved, especially the Cl 8:1 trans and cis peaks. Determine the relative retention times for each of the FAMEs in the mixed standard solution relative to C 13:0 (internal standard). Define the concentration range within which the linear range of the method will be evaluated and established. The sample extract is then injected comprising 1.0 μL sample extract onto GC column.

[0028] Further, GC/MS is conductive for analysis of fatty acids in human milk. The sample extract can be analyzed with GC/MS as follows. GC/MS conditions.—Oven: initial temperature, 140EC (hold 5 min), rate, 4.0EC/min; final temperature, 240EC; final time, 15.0 min. Zone temperatures: injector, 220EC; split ratio, 40:1; carrier gas, helium; 0.6 mL/min; interface temperature, 280EC. The equipment is preferably calibrated by autotuning prior to each series of analyses.

[0029] The relative retention times for each of the analytes are recorded from the GC chromatogram. Relative retention times are used to identify the FAMEs in the sample. Amounts of fatty acids are calculated from peak areas according to standards.

Example 3

[0030] Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass spectrometry that uses an inductively coupled plasma to ionize the sample. This is the preferred analytical technique for detecting metals and some non-metals in liquid samples present in low concentrations. This is the method of analysis used for calcium, iron, cyanocobalamin, heavy metals, and environmental toxins for example. In an embodiment of the invention, a method comprises the steps of:

[0031] a) Providing 1 ml of prepared milk sample. b) Homogenizing the prepared milk sample, after heating the sample to 40 degrees C. with inversion and vortexing. c) Digesting the homogenized sample by mixing with about 60-70% HNO3 and about 30% H2O2 in a 1:1 ratio. d) Holding mixture at a temperature of about 195° C. for a period of 20 to 45 minutes. f) cooling the mixture to room temperature and diluting the mixture to make a final volume of 50 ml with deionized water for analysis. g) Establishing a calibration curve using a standard solution with dwell time set at 50 milliseconds and thirty sweeps and three replicated with background correction are completed. h) Quantifying the signal intensities detected for each analyte and comparing the signal intensities to a calibration curve for that analyte to determine the concentration of each analyte.

Example 4

[0032] An alternate embodiment of the invention comprises preparation of a sample for ICP-MS or ICP-OES to determine concentrations of analytes including Vitamin A, Vitamin D and Vitamin E comprising the steps of: a) Adding 20 ml of prepared milk sample to a test tube b) Homogenizing the prepared milk sample, with inversion, vortexing and/or sonicating. c) Mixing 20 ml of the sample with: about 50 to 70 ml of 95% ethanol, about 10-30 ml of 50% (weight/volume) Kolliphor EL (KOL), about 40 to 60 mg of hydroquinone, and about 0.5 to 1.5 ml of 12% (weight/volume) sodium sulfide in water d) Saponifying the sample by heating the sample e) Extracting one or more analytes in the sample by: mixing the sample with a 1:1 ratio of petroleum ether and diethyl ether f) Washing the sample [with ethanol] and evaporating the solvent with a rotovap within the sample to produce a residue of solutes. g) A calibration curve is established using a standard solution with a dwell time set at 50 ms are completed. h) Quantifying an amount of one or more analytes in the sample using Liquid Chromatography with tandem mass spectrometry. Quantifying comprises quantifying the signal intensities detected for each analyte and comparing the signal intensities to a calibration curve for that analyte to determine the concentration of each analyte.

[0033] It is a further aspect of the present invention to provide a comprehensive, personalized analysis of breast milk composition, as well as individual dietary and supplement recommendations based on the results of the analysis.

[0034] Aspects of the present invention include a method of analyzing breast milk for a panel of analytes, comprising a) collecting a sample comprising breast milk from the human that produced the breast milk; b) analyzing the sample for a panel of nutritional analytes comprising one or more of: calcium, iron, Vitamin A, Vitamin C, Vitamin B 12, Vitamin D, Vitamin E, Vitamin K 1, Vitamin K2, Vitamin B1, Vitamin B2, vitamin B3, Vitamin B6, choline, folate, phosphorus, zinc, selenium, magnesium, cyanocobalamin, and environmental/toxic metals; aluminum, silicon, platinum, copper, cadmium, Lead, Mercury, Choline, Nickel, Beryllium, Boron and Hexavalent chromium. The panel of analytes can further comprise one or more fatty acids e.g., docosahexaenoic acid (DHA), arachidonic acid (ARA), Lactic Acid (LA), Eicosatetraenoic acid (EPA), and alpha-Linolenic acid (ALA), and c) providing the human with the results of the analyzed sample, wherein the results comprise a concentration of each analyte present in the sample. If the measured concentration of a nutrient analyte is in the panel of analytes is below guidance concentration for the nutrient analyte, then the human's breast milk has a deficiency of the analyte and if the measured concentration of a nutrient analyte in the panel of analytes is above a guidance concentration for the nutrient analyte, then the human's breast milk has an elevated level of the nutrient analyte. Guidance concentrations for nutrient analytes can comprise a concentration range.

[0035] Aspects of the present invention include methods of screening breast milk for a panel of nutrient analytes associated with diet, comprising: a) collecting a sample comprising breast milk from the human that produced the breast milk; b) analyzing the sample for concentration of one or more nutrient analytes; c) analyzing the sample for a concentration of one or more contaminant analytes; d) providing a dietary recommendation to the human based whether the measured concentration is above or below the guidance concentration for each nutrient analyte and each contaminant analyte. In an embodiment of the invention, nutrient analytes comprise one or more of: calcium, iron, Vitamin A, Vitamin C, Vitamin B 12, Vitamin D, Vitamin E, Vitamin K 1, Vitamin K2, Vitamin B 1, Vitamin B2, vitamin B3, Vitamin B6, thiamine, choline, folate, phosphorus, zinc, selenium, magnesium, cyanocobalamin. The nutrient analytes can further comprise a panel of analytes comprising one or more fatty acids e.g., docosahexaenoic acid (DHA), arachidonic acid (ARA), Lactic Acid (LA), Eicosatetraenoic acid (EPA), and alpha-Linolenic acid (ALA). Guidance concentration ranges have been discovered and Table 1 provides guidance concentrations for nutrient analytes.

TABLE-US-00001 TABLE 1 Guidance Concentrations of Nutrient Analytes. Guidance Guidance concentration concentration Nutrient Analyte lower range upper range Calcium bound to 5 mg/dL 45 mg/dL protein Ionized calcium 155 mg/dl 300 mg/dL Iron 0.01 mg/L 1 mg/L Vitamin A 229 μg/L 1090 μg/L Vitamin C 10 mg/L 158 mg/L Vitamin D 0.90 nM/L 1.42 nM/L Vitamin B 12 0.1 μg/L 2 μg/L Vitamin E 5 umol/L 100 umol/L Vitamin K1 0.5 umol/L 100 umol/L Vitamin K2 0.5 umol/L 100 umol/L Vitamin B1 (thiamine) 0.05 mg/L 200 mg/L Vitamin B2 (riboflavin) 10 ug/L 1000 ug/L Vitamin B3 (niacin) 50 ug/L 1000 ug/L Vitamin B6 0.01 mg/L 100 mg/L Choline 50 umol/L 1000 umol/L Vitamin B9 (Folate) 8-120 ug/L 8-120 ug/L Phosphorus 0.0001 mg/L 10 mg/L Zinc 0.001 mg/L 50 mg/L Magnesium 0.001 mg/L 50 mg/L Cyanocobalamin 0.0001 mg/L 10 mg/L DHA 0.01 to 0.5% of total fatty acids, Analyte is linoleic 3000 nmol/ml 8000 nmol/ml acid (LA) Arachidonic acid (ARA) 3000 nmol/ml 8000 nmol/ml Alpha-linoleic 350 nmol/L 800 nmol/ml acid (ALA)

[0036] Guidance concentrations of contaminant analytes have been established for environmental toxins and toxic metals, such as but not limited to aluminum, silicon, platinum, copper, cadmium, Lead, Mercury, arsenic, Nickel, Beryllium, Boron and Hexavalent chromium

TABLE-US-00002 TABLE 2 Guidance Concentrations of Contaminant Analytes. Lower Guidance Guidance Detection Contaminant concentration concentration Limit of Analyte lower range upper range ICP-MS Aluminum 0.0001 mg/L 0.05 mg/L 1 ppT Silicon 0.0001 mg/L 4 mg/L 1 ppT Platinum 0.0001 mg/L 0.003 mg/L 1 ppT Copper 0.0001 mg/L 0.05 mg/L 1 ppT Cadmium is 0.005 mg/L to 0.2 mg/L 1 ppT Lead 0.0001 mg/L 0.015 mg/L 1 ppT Mercury 0.0001 mg/L 0.002 mg/L 1 ppT Arsenic is 0.0001 to 0.01 mg/L 0.01 mg/L 1 ppT Nickel 0.0001 to 0.05 mg/L 0.05 mg/L 1 ppT Beryllium from 0.0001 to 0.05 mg/L 0.05 mg/L 1 ppT Boron 0.0001 to 0.05 mg/L 0.05 mg/L 1 ppT Chromium 0.0001 to 0.05 mg/L 0.05 g/L 1 ppT

[0037] It has been discovered through analysis, that the nutritional content of breast milk varies widely between people and over time in the same person. We have found that collecting samples through a 24-hour period from the same individual throughout various manual or machine expression methods provides a representative sample. Further, maternal diet affects the analytes including but not limited to analytes listed in Table 1 and Table 2.

[0038] Lactating mothers can receive their analytical results with personalized dietary recommendations to increase the concentration of vitamins and minerals and decrease the concentration of toxins in their breast milk.

[0039] In some embodiments, the method further comprises re-testing the human's breast milk by repeating steps of the method every 1-8 weeks for the duration of lactation. Re-testing the human's breast milk periodically can improve the overall health and diet of the human. Re-testing the human's breast milk periodically and providing dietary recommendations is configured to increase the human's chances of prolonging breastfeeding. Re-testing the human's breast milk periodically and providing dietary/lifestyle recommendations is configured to improve the quality of the human's breast milk. Providing dietary recommendations is configured to decrease the level of anxiety and/or stress and provide feedback to the human associated with breastfeeding. Testing under the method of the invention can be repeated to provide insight as to the effect of dietary changes and make further dietary recommendations. The process of testing and providing feedback can continue through the lactation period of the nursing individual.

[0040] Reference ranges for each micronutrient are shown in Table 1 and the breastfeeding mother can be informed of her results as compared to the average. Lifestyle and dietary recommendations can be reviewed by a team of physicians to provide recommendations on how to improve the concentrations of nutrient analytes in human milk. It has been discovered that maternal diet impacts nutrient concentrations in breast milk, and a mother has the ability to change her diet to affect the concentration of said nutrient concentrations. It has been discovered that nutrient concentrations in breast milk is variable and information about the composition of an individual's milk is useful for making dietary decisions and nursing decisions.

[0041] The operations described herein can be performed in any sensible order. Any operations not required for proper operation can be optional. Further, all methods described herein can also be stored on a computer readable storage to control a computer. The many features and advantages of the invention are apparent from the detailed specification and, thus, it is intended by the appended claims to cover all such features and advantages of the invention that fall within the true spirit and scope of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.