TITRATION OF THERAPY THROUGH ASSAY OF CHOLESTEROL IN SKIN BY MASS SPECTROSCOPY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to United States Provisional Patent

Application No. 61/675,925 filed July 26, 2012 and United States Provisional Patent Application No. 61/767,827 filed February 22, 2013.

The content of the above patent applications are hereby expressly incorporated by reference into the detailed description hereof.

FIELD OF THE INVENTION

The present invention relates to the field of the measurement of biomarkers in skin samples for assessment of health risk in individuals and titration of a therapy. More particularly, the present invention provides a non-invasive method of titrating therapy through measuring biomarkers in skin samples more precisely, measuring skin samples sampled from the skin surface through calibration using a molecular marker also contained in the skin sample and using said measurements to assess need for therapy, response to interventional therapy, and titrate said therapy. BACKGROUND

Skin is the largest organ in the body and the surface layer of dead cells is constantly being exfoliated making it a non-invasive source of biomarkers. Early detection of disease or risk of developing disease is seen as a crucial step for effective management. Effective management of some conditions, such as elevated cholesterol levels, requires careful monitoring and possible titration of therapies through effective observation over time of biomarkers.

Unfortunately, most diagnostic tests and titration methods require invasive samplings such as blood or collection of urine which requires skilled personnel, careful storage, and in the specific case of titration multiple painful samplings. Simple extraction of skin samples through various means and follow up assays would alleviate many expensive burdens on the health care system and various burdens on the patient. These skin samples could be analyzed immediately or sent to a lab for large scale processing, allowing for quick generation of information needed for effective adjustment of therapies for patients. Excessive delay in titrating statin therapy to the optimal dose may lead to an increased risk of atherosclerosis- related events in high risk patients (Friend, A.S., et al. Effect of Time To Titration to Maximum Dose of Statins on CV Outcomes: A Retrospective Analysis in High Risk Patients. Am J Cardiovasc Drugs. 2009;9(2): 103-7.). Since the outer layer of skin is dead and can be removed with ease, it would serve as an ideal source of non-invasively obtained biological specimens which require neither skilled personnel to sample nor special conditions to maintain stability

The stratum corneum contains an assortment of proteins, lipids, and other macromoleculres. More specifically, skin contains cholesterol which has been shown to be a valuable biomarker for cardiovascular disease risk assessment. Numerous studies have shown that atherosclerosis and its complications, such as heart attacks and strokes, are major causes of morbidity and mortality in almost all countries of the world.

Cost effective prevention of atherosclerosis or treatments for elevated cholesterol requires the identification of individuals at risk and effective therapy through proper titration, respectively. A widely used method for identifying individuals at risk of having atherosclerosis is based on the measurement of total cholesterol levels in venous blood plasma (Consensus Conference on lowering Blood Cholesterol to Prevent Heart Disease, JAMA, 1985, 253, pg. 2080). In fact serum or plasma cholesterol is the current standard for determination of dyslipidemia; patients are considered to be at high-risk if their cholesterol level is over 240 mg/dL and there have been recent moves to lower this threshold level to lower values.

Statins are the most commonly used medications for the treatment of dysipidemia; used to lower cholesterol levels in patients through inhibition of HMG-CoA reductase. Unfortunately statins are frequently not taken and as a result National Cholesterol Education Program (NCEP) target goals are not achieved (Foley, K. A., et al. Effectiveness of statin titration on LDL-Cholesterol Goal Attainment in Patients At High Risk of Atherogenic Events. Am J Cardiol. 2003 ;92(1): 79-81; Brown A.S., et al. Treating Patients With Documented

Atherosclerosis to NCEP -Recommended LDL-Cholesterol Goals With Atorvastatin, Fluvastatin, Lovastatin, and Simvastatin. J Am Coll Cardiol. 1998;32(3):665-72.). Upward titration of drugs, such as statins, is an obvious, cost effective, and simple therapeutic step to increase NCEP goal attainment. One reason why patients may fail to achieve LDL cholesterol goals with statin treatment is because statins are not appropriately titrated (Andrews T. C, et al. Achieving and Maintaining National Cholesterol Education Program LDL-Cholesterol Goals With 5 Statins. Am J Med. 2001;111(3): 185-91.; Vicari et al., Use of Simvastatin Treatment in Patients With Combined Hyperlipidemia in Clinical Practice. Arch Fam Med. 2000;9(9):898-905.)

Traditional methods for titration of therapy are invasive and may require fasting. This generally requires the patient to attend a blood sampling facility in order to provide the sample. The time required to provide the sample and the requirement for a blood sample are thought to contribute to the generally poor overall poor statin medication compliance.

Additionally, the sampling poses a risk of infection, bruising, and personnel such as trained phlebotomists are required to obtain the sample which increases the cost of the process.

Significant levels of cholesterol occur in tissues as well as in plasma and it has been shown that tissue cholesterol plays a leading role in development of atherosclerosis. Skin cholesterol has been shown to be significantly associated with positive stress tests, angiographic coronary artery disease (CAD), and prior events. Additional studies indicate that skin cholesterol is also associated with circulating inflammatory markers and coronary artery calcium and may therefore serve as a useful marker of subclinical atherosclerosis. For example, cholesterol was extracted from lyophilized skin samples and measured using traditional chemical and biochemical techniques. (Nikitin Y. P., Gordienko I. A. Dolgov A. V., Filimonova T. A. "Cholesterol content in the skin and its correlation with lipid quotient in the serum in normals and in patients with ischemic cardiac disease", Cardiology, 1987, II, No. 10, P.48-51). While useful, this method is too complicated and painful to be employed for large scale population screening.

Determination of the cholesterol content in the skin gives a measure of the extent of atherosclerosis and can be obtained through standard laboratory analysis of skin biopsy specimens. Further, these cholesterol measurements can give a reading as to a patient's response to a therapy and allow titration of said therapy. However, there is considerable pain involved in taking a skin sample and a risk of infection at the sampling site. In addition, this method has other disadvantages because the thick skin specimens incorporate several skin layers, including the outermost horny layer (stratum corneum), epidermis and dermis. Since the dermal layer is highly vascularised, skin biopsy samples contain blood vessels and blood elements. They may also contain sweat and sebaceous glands and the secretions contained therein. Additionally, subcutaneous fat is located directly under the derma and may also contaminate the specimens. Therefore, skin biopsy specimens are heterogeneous and their analysis may give false data on cholesterol content in the skin. U.S. Patent No. 5,489,510 describes a non-invasive method for the visual identification of cholesterol on skin using a reagent having a specific cholesterol binding component in combination with a reagent having an indicator component to provide a visual color change corresponding to the presence of the component bound to cholesterol of the skin. The procedure is done directly on the palmar skin and while it is quick and simple, it requires all individuals to be tested to be present at a doctor's office or clinic where the test is conducted.

U.S. Patent No. 7,238,494 describes a method for measure skin cholesterol from skin samples removed by tape stripping using a cholesterol binding component and an indicator component to provide a visual change in colour corresponding to the presence of the component bound to cholesterol in the skin sample. Use of a tape stripping device for skin sampling is a solution to the problem presented with biopsies but immediate biochemical follow-up assays to determine analyte levels can be skewed based on differential skin amounts obtained through multiple samplings through a tape device. An alternative analyte measurement technique, mass spectroscopy, can be of use in determining multiple constituents of a biological sample. Molar ratios of the lipids, including cholesterol, in stratum corneum have been determined on samples obtained by direct, solvent extracted skin (Norlen L., et al. J. Invest. Dermatology 72- 77, 112, 1999). High performance liquid chromatography (HPLC) and gas liquid

chromatography (GC) in conjunction with mass spectroscopy were used to separate and analyze the lipids. Together these techniques can obtain a quicker and more easily obtained picture of a patients need for therapy, need for a change in type of therapy, or need for an alteration in therapy amounts through co-measurement of a reference analyte on sampled skin.

Summary of the Invention

The present inventors have found a biomarker and method suitable for not only non- invasively detecting at risk individuals but also non-invasively measuring results of therapy for titration of said therapy.

The method of measuring skin analyte obtains a skin sample through a quick, painless, and non-invasive method. The method also quantifies the quantity of skin cells removed. Thus, the skin analyte readings are calibrated with a known standard entity and a more accurate biomarker level determined. Further, the method of titrating therapy does not inconvenience the patient through the need for fasting, scheduling of doctor appointments, and the pain of invasive sampling. It is therefore an object of the present invention to overcome present limitations and to provide a non-invasive and accurate measurement of analyte obtained from skin samples removed, preferably by a tape stripping device for the purposes of monitoring risk of CVD and conditions, such as high cholesterol, and titrating therapies for said conditions. In one aspect of the invention, there is provided a method of measuring skin analyte which comprises the steps of: a) Obtaining a representative sample of the skin from the selected area by removal of skin cells from an individual, the individual preferably being considered for a therapy, on a therapy for a risk of CVD and/or a condition such as high cholesterol or possibly a need of a therapy for a risk of CVD and/or a condition such as high cholesterol. b) Analytes, such as skin cholesterol, from the skin cells removed are extracted with solvents appropriate for mass spectroscopy c) The extracted sample is analyzed by mass spectroscopy d) Comparison of the analyte in the skin sample to a reference analyte in the same skin sample is made e) A calibrated amount of skin analyte is determined and used to gauge the health risk status of the individual. f) The calibrated amount of skin analyte is used to assess the individuals response to a therapy or need for a therapy g) Therapy is determined to be needed, sufficient, or is altered based on the

calibrated amount of analyte. h) The steps a) through g) are repeated at subsequent time points in the future and results of analyte levels against time is plotted for visual comparison. i) Summary tables can be provided for all baseline variables, primary endpoint variables, secondary end point variables, and subsequent end point variables. The skin samples in the aforesaid method are collected from methods selected from scraping or tape stripping.

Also, in the aforesaid method a pre-determined amoimt of solvent suitable for mass spectroscopy is applied to the skin sample. In the method for the invention, the separation and purification of the lipids and/or proteins can be carried out through chromatography, in one aspect this is carried out through HPLC, in another aspect this is carried out through GC. The sample molecules to be analyzed are ionized from the sample to produce one or more detectable ions for mass spectroscopy. The amount of ion(s) of the analyte can be detected by mass spectroscopy. The amount of ion(s) detected are then used to measure the amount of analyte in said sample.

In one aspect of the method the analyte is cholesterol.

The calibration measurement is used with a view to combining a reference analyte, preferably total protein or a protein such as collagen, keratin, elastin, or

glycosaminoglycans and/or ceramide measurement obtained, representative of the amount of skin sample extracted, with a corresponding analyte measurement to identify individual's current health status and a need or response to therapies for a health status.

Skin samplings and analyte measurements can be taken over a prolonged time span and can be plotted to determine a subjects need for therapy or therapies, response to a therapy or therapies, need for alterations in a therapy or therapies, addition of a therapy to already existing therapy or therapies, and ending of a therapy or therapies.

Brief Description of Figure

Figure 1 is a graph showing the correlation between number of samplings and quantity of cholesterol in the sample.

Detailed Description A more accurate measurement of biomarkers/analytes found in the skin is taught through calibrating the amount of said biomarker/analyte present with the amount of skin assayed and using said calibrated amount of biomarker/analyte to assess need of a therapy, effectiveness of a therapy, and possibly titrate said therapy. The amount of skin assayed is determined through quantification of a reference analyte such as total protein but preferably ceramides and/or proteins such as collagen, keratin, elastin, or

glycosaminoglycans found in said skin sample. The skin sample can be obtained in any manner but is preferably from the stratum corneum and preferably through use of an adhesive medical tape. The skin cells can be obtained from any part of the skin, but the most suitable part is the surface of a palm because the palm does not have sebaceous glands whose secretions contains cholesterol that may affect results for certain aspects of the method, and particularly those aspects involving measuring cholesterol. Additionally the skin on the palm is readily accessible for sampling.

It is desirable to obtain uniform amounts of skin cells for sampling to get accurate readings for biomarkers. Skin sampling such as skin taping and skin scraping will achieve adequate amounts of skin cells for measurement of biomarkers but may lead to variance in these markers from individual to individual due to variations in skin cell amounts obtained. Even with saturation of medical adhesive strips with skin cells through multiple applications to the skin, there may exist variance from individual to individual in the amount of skin cells analyzed. This problem would be compounded when using simpler sampling methods, where any quantification of skin cells without the use of additional assays would be impossible. The total amount of biomarker analyzed and therefore, present, in the skin is directly related to the amount of skin sample obtained. Therefore the need to normalize readings is necessary for adequate reliability of said readings.

Medical adhesive tape for skin sampling, as that in the present applicant's issued patent U.S. Patent No. 7,238,494, the contents of the entirety of which are hereby incorporated as a reference, can be applied to obtain a number of skin cells from the stratum corneum. A number of skin cells from a number of individuals can be measured for various analytes, preferably cholesterol, to assess the individuals health risk, or lack thereof, of ailments, preferably atherosclerosis and need of a therapy or titration of said therapy

Obtaining consistently normalized skin samples from individuals or repeated samples from the same individual is accomplished by the following steps. In one embodiment, the skin sample is taken by applying a pre-determined sized adhesive repeatedly to the skin such that it becomes saturated with skin and is no longer sticky. Ten applications are done to ensure saturation. Alternatively, in another aspect of the method, obtaining consistent sized skin samples from individuals (or repeated samples from the same individual) is accomplished by applying an abrasive to the skin and skin cells are collected. After obtaining a representative sample of skin from the individual the sample is then extracted with an appropriate solvent for mass spectroscopy. The methods utilize liquid chromatography (LC), most preferably HPLC, or GC, to perform a purification of selected analytes from the skin sample, and combine this purification with mass spectrometry (MS), thereby providing a high-throughput assay system for detecting and quantifying multiple analytes in a test sample. The function and advantage of this method is that it co-currently compares an analyte of interest with a reference analyte to obtain a better and more accurate relationship between skin sample and analyte measured by also indirectly measuring skin sampled. Selection of a reference analyte is made. The outer horny -layer of skin (stratum corneum) consists largely of protein-enriched corneocytes surrounded by a lipid mixture that includes cholesterol. Structurally, this is often depicted as a "brick and mortar" model with the corneocytes representing the bricks and the surrounding lipids representing the mortar (P.M. Elias, J Invest Dermatol. 1983, 80, 44S-9S). The amount of protein in the stratum corneum is relatively constant between different individuals; therefore protein in the skin sample removed can provide an indirect measure of the amount of skin removed. Ceramides can alternatively be used, being relatively constant between different individuals. By measuring proteins such as collagen, keratin, elastin, or glycosaminoglycans and/or ceramides, and therefore, the amount of skin removed, and using this amount or measurement as a calibration amount, a proper quantification of skin analyte can be obtained. This amount can be a measurement of skin analyte per unit of protein level, ceramide level and, if desired, skin analyte per unit amount of skin.

For patients with a condition, such as but not limited to high cholesterol, before commencement of a therapy or during a therapy, the calibrated amount of analyte can be used to assess one's initial need or adjustment of therapy. For patients beginning a therapy, where therapy may be but is not limited to pharmacological therapy or lifestyle changes, the initial amount of calibrated analyte from the skin surface will serve to dictate the initial amounts of therapy. For patients on continued therapy the amount of calibrated analyte from the skin surface can be used to dictate adjustments in therapy. These continued samplings can take place at multiple time points during the course of one's therapy to determine the ongoing need for therapy, changes in levels of therapy, additions of therapy, or switches in therapy. Further, the amount of calibrated analyte from the skin surface can be used to determine a need for a change in therapy and the levels of new therapy required. Example 1-Skin Cholesterol Measurements and Calibration of Measurements

Cholesterol Confirmation by LCMSMS Purpose: To provide a method specific testing parameters for LC/MS/MS for the analysis of cholesterol in human skin cells.

Clinical/Forensic Significance: Cholesterol is a steroid with a secondary hydroxyl group in the C3 position. It is synthesized in many types of tissue, particularly in the liver and intestinal wall. Approximately three quarters of cholesterol is newly synthesized and a quarter originates from dietary intake. Cholesterol assays are used for screening for atherosclerotic risk and in the diagnosis and treatment of elevated cholesterol levels and lipid/lipoprotein metabolic disorder. Principle: Deuterated cholesterol (cholesterol-D6) is added as an internal standard to skin cells. The cells are crashed and extracted with an organic solvent. The extracted solution is then injected into the LC/MS/MS system and analyzed.

Specimen: Human cells collected by tape

Reagents:

1. Acetonitrile (JT Baker, 9017-03 2. Methanol (Fisher, E2362)

3. Anhydrous Ethyl Alcohol (Commercial Alcohols Inc., 432526) Materials:

1. Hole Puncher 6mm

2. Disposable Culture Tubes 10 x 75 mm (Kimble)

3. Flat Top Stopper Caps for 10mm tubes (Evergreen Scientific)

4. Vortex mixer (Fisher)

5. Shaker (Eberbach Corp)

6. Ultrasonic Bath with heater (VWR)

7. IOOUL polysterene autosampler vials with glass inserts (Agilent) 8. Vial snap it seal caps (Agilent)

Calibrators:

1. Cholesterol Calibrator: (TRC Inc, Cat No. C432501) Concentration = lOg

2. Cholesterol - d6: (TRC Inc, Cat. No. D229455)

Concentration = 25mg

3. Cholesterol Stock calibrator:

Concentration = 100000 ng/niL

Dissolve lmg of Cholesterol calibrator in 10 mL with methanol. 4. C18 Ceramide Calibrator: (TRC Inc, Cat No. C263050)

Concentration = 5mg

5. C18 Ceramide-13C2,d2: (TRC Inc. Cat No. C2603052) Concentration = lmg

6. C18 Ceramide Stock Calibrator:

Concentration = 100000 ng/mL

Dissolve lmg of C18 Ceramide calibrator in 10 mL with methanol. 7. C 18 Ceramide Intermediate Calibrator:

Concentration = 1000 ng/mL

Dissolve 100μL of C18 Ceramide stock calibrator in 10 mL with methanol.

8. Working Calibrator:

Concentration = 2000 ng/mL Cholesterol; lOng/mL CI 8 Ceramide

Dilute 200 uL of Cholesterol stock calibrator and 100 μL in 10 mL with methanol.

9. Cholesterol-d6 Stock Internal Standard:

Concentration = 100000 ng/mL

Dissolve lmg of Cholesterol-d6 internal standard in lOmL with ethanol.

10. C18 Ceramide-13C2,D2 Stock Internal Standard: Concentration = 100000 ng/niL

Dilute lmg C18 Ceramide- 13C2,D2 Internal Standard in lOmL with ethanol.

11. C18 Ceramide-13C2,D2 Intermediate Internal Standard:

Concentration = 1000 ng/mL

Dilute 100 μΐ. C18 Ceramide-13C2,D2 stock Internal Standard in lOmL with ethanol.

12. Working Internal Standard:

Concentration = 3000 ng/mL Cholesterol-d6; 30 ng/mL C18 Ceramide-13C2D2

Dilute 300 uL Cholesterol-d6 stock internal standard and 300 uL C18 Ceramide- 13C2,D2 Internal Standard in lOmL with ethanol.

Skin sampling procedure: Skin samples are taken from the subject using a medical grade adhesive (e.g. medical grade adhesive manufactured by 3M) attached to a sampling device. The adhesive is applied with pressure to the palm of the subject's hand, with each sampling taking from a different part of the patients palm.

Sample Preparation:

Remove tape collection device from storage bag.

Punch out spot from the center of the collection device using the 6mm punch, Prepare blank punches for calibrators (Blk, CAL 1, CAL 2, CAL 3) and quality controls.

Place punches (calibrators, quality controls, and duplicate samples) in 10 x 75 mm disposable culture tubes.

Add 100 μL of working internal IS to each well.

Add appropriate amount of working calibrator to each calibrators.

10 ng/mL Ceramide)

7. Add 200 μΐ, of methanol to each samples. Cap tubes.

8. Vortex for 30 sec.

9. Sonicate the capped tubes for 30 min at 60 °C.

10. Place the tubes in the shaker and shake for 5 minutes.

11. Remove tubes. Transfer samples into autosampler vials.

12. Inject 20 μΐ, into the LC/MS/MS.

Instrument Parameters:

LCMSMS System: Thermo TSQ Quantum Ultra

Scan Type: APCI

Polarity: Positive

Scan Mode: SRM

Ion Source: Turbo Spray

Resolution Ql : 0.7 (Unit)

Resolution Q3: 0.7 (Unit)

Discharge Current: 4.00

Vaporizer Temp: 425.00

Sheath Gas: 25.00

Ion Sweep Gas: 0.00

Aux Gas: 5.00

Chromatographic Parameters:

HPLC Pump: Thermo Accela

AutoSampler: Thermo PAL/CTC

Mobile Phase: A: water

B: Acetonitrile

Elution: Gradient

Flow Rate: 700uL/min

Column: Phenomenex Synergi 4μ POLAR RP 80A (250 x 2.00 mm 4 micron)

Acquisition Time: 6 min

Injection volume: 25 uL

Retention Time: 3.33 min Cholesterol; 3.25 min Ceramide

Mass Transitions:

Cholesterol-d6

375.008 - 109.511

375.008 - 152.046

375.008 - 166.996

Cholesterol

369.005 - 105.094

369.005 - 147.019

369.005 - 161.086 (quant)

CI 8 Ceramide

566.060 - 251.980

566.060 - 264.072

566.060 - 548.377

C18Ceramide-13C2,d2

570.065 - 268.086

570.065 - 285.964

570.065 - 552.358 (quant)

Data Interpretation and Calculations:

Quantitative results are calculated by linear regression using the Quantitation Module of the LC Quant software program. All the transitions are calibrated in this manner to evaluate ion ratios, though only the quantitation ion is used for quantitation.

Performance criteria: Correlation coefficient of the calibration curve (r ² ) must be 0.99 or higher for all ions.

The calibrators (CAL 1, CAL 2, CAL 3) quantitative values must be within ±20% of their target values. The quantitative value for the blank should be below the LOD (limit of detection) For compound identification, retention time of the analyte must agree with the retention time of the corresponding internal standard within ±5%. Furthermore, the calculated concentration of the ions must be within ±20%.

If the quantitative value of a sample is over the linear range and the retention times are out of range, make the appropriate dilution (diluted concentration should be above the lowest calibrator) and reinject.

Conditions of Procedure:

• Limit of detection (LOD): 100 ng/mL Cholesterol; 0.5 ng/mL Ceramide

• Limit of Quantitation (LOQ): 100 ng/mL Cholesterol; 0.5 ng/mL Ceramide

• Upper Limit of Linearity (ULOL): 2000 ng/mL Cholesterol; 10 ng/mL Ceramide

· Carryover: 2000 ng/mL Cholesterol; 10 ng/mL Ceramide

Example 2- C16 Ceramide as a Skin Sample Calibrator

Skin sampling with adhesive tape may result in different amounts of sample depending on the sampling technique, the type of skin the individual has (e.g., dry or moist), and environmental conditions. When measuring analytes in the sampled skin sample quantity variation will confound sample comparison within and between individuals. The co-determination of a common skin lipid, ceramide, can be used to calibrate the amount of sample and thus allow for more accurate comparisons between skin samples.

Method:

Variable amounts of skin were deliberately acquired from an individual by sampling the two palms of an individual from one to 20 times.

Cholesterol and CI 6 Ceramide were determined from the various samples by mass spectroscopy

Results: Figure 1 shows that increasing number of samplings increases the quantity of cholesterol in the sample as more skin is sampled with more applications of the sampling device. By adjusting the cholesterol value using the amount of ceramide 16 in the sample the concentration of skin in the sample becomes consistent as would be expected because all of the samples are from one individual.

Conclusions:

Adjusting cholesterol values with ceramide C16 to compensate for variable sampling improves sample to sample consistency with respect to cholesterol concentration.

Example 3 - Titration of therapy using calibrated skin cholesterol levels

Implimentation and monitoring of lipid lowering requires titration of lipid lowering medication to ensure the target lower lipid levels are being achieved at the minimal required medication dose. This generally requires multiple blood samplings at intervals from the subject which can be a deterrence for proper follow-up. Skin cholesterol through skin sampling can act as a non-invasive analyte to measure response to lipid lowering and modification of medication dosage if necessary. Additionally, calibrating the skin cholesterol measurement with a control analyte from the same sample can yield a more accurate overall measurement since it adjusts for amount of sample. This method may be applicable to any medications or diets or behavioural changes that lower serum lipids and/or lower risk of cardiovascular disease. The reduction of skin cholesterol levels in response to such interventions may provide added incentive to continue medications or regimens that are risk reducing. Titration of statin therapy using calibrated skin cholesterol measurements: Statin therapy treatment are prescribed at a small dosage (for example, Simvastatin; 5mg daily, orally injested) after initial skin sampling and analyte measurement. The subject will return at a second time point (for example, 30 days since initial measurements) to once again have skin analytes measured and a calibrated skin cholesterol measurement deduced. The dosage of statin is adjusted to either increase or decrease based upon changes in calibrated skin cholesterol levels. This process of measuring calibrated skin cholesterol levels and adjusting dosages of statin will continue until an ideal dosage and skin cholesterol measurement is found. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

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