CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of priority to U.S. Patent Application No. 62/076,050 filed on Nov. 6, 2014, the disclosure of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present application generally relates to a method and device for screening vitamin D (Vit-D) insufficiency. In particular, the present application relates to a method and device for screening vitamin D insufficiency based on skin colourimetry.

BACKGROUND

Vit-D is important for skeletal growth and bone mineralization during pubertal growth. The best indicator for Vit-D status is serum 25(OH)Vit-D level with Vit-D insufficiency being diagnosed when serum 25(OH)Vit-Dâ‰¦50 nmol/L(1). Vit-D insufficiency has been reported to be highly prevalent across all age groups in different countries and geographical regions (2). In spite of this, the potentially treatable condition remains under-recognized resulting in perpetuation of a global epidemic in Vit-D insufficiency (3) and the adolescent group is no exception. Apart from inadequate awareness for this health problem and the absence of symptoms in its early stage, the lack of screening tests can be the culprit. Serum 25(OH)Vit-D assay requires blood taking which is costly, not readily available and is not suitable as a screening tool for the young population. Neither are the sunlight exposure questionnaire (4-6) nor the personal UV dosimeter (7). Predictive models using patient's demographic and lifestyle characteristics have been reported but were confined to adults and the elderly (4, 8-11).

There remains in the art a long-awaited quest for an effective screening tool for Vit-D insufficiency.

SUMMARY

The present application provides a non-invasive method and device for screening of Vit-D insufficiency.

In a first aspect disclosed herein, there provided is method for establishing a process for screening vitamin D (Vit-D) insufficiency in a population of interest, comprising

determining a Vit-D status of a cohort of subjects of the population by measuring serum 25(OH)Vit-D level, wherein serum 25(OH)Vit-D levelâ‰¦50 nmol/L indicates Vit-D insufficiency, and serum 25(OH)Vit-D level>50 nmol/L indicates Vit-D sufficiency, and wherein Vit-D insufficiency status is designated a value of â€œ1â€ and Vit-D sufficiency status is designated a value of â€œ0â€ ;

obtaining a set of factors including constitutive skin melanin pigmentation measurement (Vf) and facultative skin melanin pigmentation measurement (Vh) in an individual subject, and one selected from the group consisting of: an age, a season, a gender, a body weight (BW), a standing height (SH), a physical activity level (PAL), a daily dietary Vit-D intake (Dvd), a daily dietary calcium intake (Dca), or any combination thereof in the individual subject, or transformed formats of the factors, wherein Vf is measured using skin colourimetry on an instep area, an inner aspect or a sole of a foot, or a heel of the individual subject, Vh is measured using skin colourimetry on a dorsum of a hand, fingers, a thumb or a wrist of the individual subject, the age, the BW, the SH, the PAL, the Dvd or the Dca is determined from the individual subject, the seasons are differentially designated pre-set values, and the genders are differentially designated pre-set values, and

carrying out a logistic regression analysis by using a Vit-D status as a dependent variable, and the factors or the transformed formats of the factors as independent variables, thereby obtaining a function as shown below with regression coefficients for each of the independent variables:

$\log_{e} î¢ (\frac{Pred}{1 - Pred}) = Î± + {âˆ‘}_{1}^{k} î¢ž î¢ž {Î²}_{i} î¢ž x_{i},$

wherein Î± is a constant term,

Î²_iis a regression coefficient for an independent variable x_i,

x_irepresents the individual independent variables,

k represents a total number of the independent variables,

Pred is a predicted probability used for determining a screening result of either Vit-D insufficiency or Vit-D sufficiency according to a pre-set cut-off threshold of Pred.

In some embodiments, Vf and/or Vh is measured using Individual Typology Angle (ITA) based on a skin colourimetry system, e.g. L*a*b* colour system.

In some embodiments, in season factors, summer is designated value â€œ1â€ and winter is designated value â€œ2â€ .

In some embodiments, in gender factors, male is designated value â€œ1â€ and female is designated value â€œ2â€ .

In some embodiments, the population of interest is an Asian population.

In some embodiments, the population of interest is a Chinese population.

In some embodiments, the population of interest is adolescent population in

Hong Kong, and the obtained function is:

Log_e(Pred/(1âˆ’Pred))=0.072(Vh)âˆ’0.095(Vf)âˆ’0.09(season)âˆ’0.297(gender)âˆ’0.005(BW/SH²)âˆ’0.29(age)+8.055.

In some embodiments, the population of interest is female adolescent population in Hong Kong with daily dietary Vit-D intakeâ‰¦400 IU/day, and the obtained function is:

Log_e(Pred/(1âˆ’Pred))=0.131(Vh)âˆ’0.162(Vf)âˆ’0.293(season)âˆ’0.104(BW/SH²)âˆ’0.358(age)+12.997.

In some embodiments, the population of interest is adolescent population in Hong Kong, and the obtained function is:

Log_e(Pred/(1âˆ’Pred))=0.057(Vh)âˆ’0.087(Vf)âˆ’0.22(season)âˆ’0.015(gender)+0.007(BW)âˆ’0.034(SH)âˆ’0.203(age)âˆ’0.6(PAL)+13.388.

In some embodiments, the pre-set cut-off threshold of Pred is about 0.61.

In a second aspect disclosed herein, there provided is a method for screening vitamin D (Vit-D) insufficiency in a subject, comprising

providing a function for screening vitamin D (Vit-D) insufficiency in a population that the subject is from according to the method as described in the first aspect,

obtaining a set of factors in the subject as defined in the function,

introducing the set of factors into the function thereby obtaining a Pred, and

comparing the calculated Pred with a pre-set cut-off threshold of Pred, wherein the calculated Predâ‰§the pre-set cut-off threshold of Pred indicates Vit-D insufficiency, and the calculated Pred<the pre-set cut-off threshold of Pred indicates Vit-D sufficiency.

In some embodiments, the providing a function for screening vitamin D (Vit-D) insufficiency includes establishing a function for screening vitamin D (Vit-D) insufficiency in a population that the subject is from according to the method as described in the first aspect.

In a third aspect disclosed herein, there provided is a device for screening vitamin D (Vit-D) insufficiency in a subject, comprising

a measuring module configured to measure and record constitutive skin melanin pigmentation measurement (Vf) and facultative skin melanin pigmentation measurement (Vh) in the subject based on skin colourimetry, wherein Vf is measured on the instep area, the inner aspect or the sole of the foot, or the heel of the subject, and Vh is measured on the dorsum of the hand, the fingers, the thumb or the wrist of the subject,

an inputting module configured to record one or more or all of age, season, gender, body weight (BW), standing height (SH), physical activity level (PAL), daily dietary Vit-D intake (Dvd) and daily dietary calcium intake (Dca) in the subject, or transformed formats of the factors, wherein, age, BW, SH, PAL, Dvd or Dca is determined from the subject, seasons are differentially designated pre-set values, and male and female are differentially designated pre-set values, and

an processing module configured to run a function for screening vitamin D (Vit-D) insufficiency in a population that the subject is from according to the method described in the first aspect and calculate a Pred.

In some embodiments, the device further comprises a comparing module configured to compare the calculated Pred with a pre-set cut-off threshold of Pred, wherein the calculated Predâ‰§the pre-set cut-off threshold of Pred indicates Vit-D insufficiency, and the calculated Pred<the pre-set cut-off threshold of Pred indicates Vit-D sufficiency.

In some embodiments, the measuring module works based on Individual Typology Angle (ITA) measure, e.g. L*a*b* colour system.

In some embodiments, all the modules are incorporated into a skin colourimetry system, e.g. a skin colourimeter.

In a fourth aspect disclosed herein, there provided is a computer product comprising a computer readable medium storing a plurality of instructions for carrying out the method of the first and/or the second aspect.

BRIEF DESCRIPTIONS OF DRAWINGS

FIG. 1 is an exemplary skin colourimeter based on L*a*b* colour system used in some embodiments of the present application;

FIG. 2 illustrates an ROC curve for all subjects; and

FIG. 3 illustrates an ROC curve for females with daily dietary Vit-D intake<400 IU/day.

DETAILED DESCRIPTIONS

The present application is at least partially based on the conception of potential use of skin colourimetry in screening vitamin D (Vit-D) insufficiency.

Skin colourimetry can be used to measure skin melanin pigmentation. Melanin absorbs UV radiation and thus prevents UV photons from converting 7-dehydrocholesterol to previtamin D3 (the precursor for Vit-D)(3). This natural amount of melanin present in the unexposed skin is represented by the constitutive skin colour. On the other hand, the colour of skin exposed to sunlight gets darker (tanning) with greater UV exposure resulting in higher Vit-D level. This facultative skin colour is the surrogate measurement of sunlight exposure. The combined interpretation of unexposed skin colour (the constitutive skin colour) and exposed skin colour (the facultative skin colour) can be used respectively to assess dermal capability in synthesizing Vit-D and the degree of sunlight exposure(12). With recent advancement in reflectance colourimetry, non-invasive measurement of the natural constitutive skin colour and the facultative skin colour can be swiftly done with a hand-held optical device (skin colourimeter, see FIG. 1 for an example). Since local dietary sources of Vit-D are scanty (3, 13), the main source of Vit-D is from sunlight-dependent dermal synthesis the extent of which and hence the Vit-D status of subjects is supposed to be effectively assessed by skin colourimetry.

Although various usefulness of skin colourimetry has been reported in the art, no study has been conducted to evaluate its role in screening for Vit-D insufficiency. Thus, one object of the present application is to evaluate whether skin colourimetry can be used to screen for Vit-D insufficiency, and develop a screening tool based on skin colourimetry.

The inventors of the present application also contemplate a series of confounding factors which may impact the Vit-D level in a subject, and thus incorporate the factors into the method or device for screening for Vit-D insufficiency in the present application.

In a first aspect disclosed herein, there provided is method for establishing a process for screening vitamin D (Vit-D) insufficiency in a population of interest, comprising determining a Vit-D status of a cohort of subjects of the population by measuring serum 25(OH)Vit-D level, wherein serum 25(OH)Vit-D levelâ‰¦50 nmol/L indicates Vit-D insufficiency, and serum 25(OH)Vit-D level>50 nmol/L indicates Vit-D sufficiency, and wherein Vit-D insufficiency status is designated a value of â€œ1â€ and Vit-D sufficiency status is designated a value of â€œ0â€ ;

obtaining a set of factors including constitutive skin melanin pigmentation measurement (Vf) and facultative skin melanin pigmentation measurement (Vh) in an individual subject, and one selected from the group consisting of: an age, a season, a gender, a body weight (BW), a standing height (SH), a physical activity level (PAL), a daily dietary Vit-D intake (Dvd), a daily dietary calcium intake (Dca), or any combination thereof in the individual subject, or transformed formats of the factors, wherein Vf is measured using skin colourimetry on an instep area, an inner aspect or a sole of a foot, or a heel of the individual subject, Vh is measured using skin colourimetry on a dorsum of a hand, fingers, a thumb or a wrist of the individual subject, the age, the BW, the SH, the PAL, the Dvd or the Dca is determined from the individual subject, the seasons are differentially designated pre-set values, and the genders are differentially designated pre-set values, and

carrying out a logistic regression analysis by using a Vit-D status as a dependent variable, and the factors or the transformed formats of the factors as independent variables, thereby obtaining a function as shown below with regression coefficients for each of the independent variables:

$\log_{e} î¢ (\frac{Pred}{1 - Pred}) = Î± + {âˆ‘}_{1}^{k} î¢ž î¢ž {Î²}_{i} î¢ž x_{i},$

wherein Î± is a constant term,

Î²_iis a regression coefficient for an independent variable x_i,

x_irepresents the individual independent variables,

k represents a total number of the independent variables,

Pred is a predicted probability used for determining a screening result of either Vit-D insufficiency or Vit-D sufficiency according to a pre-set cut-off threshold of Pred.

The inventors of the present application contemplate the unique feature of the method (i.e. skin colourimetry based technology) and the differences of various populations in nature, e.g. place of residence, climate, skin colour, general physical constitution, etc, and develop a method that can be applied to any population of interest on which the screening is to be conducted.

In general, the method is based on analysis of skin colourimetry measurements and other relevant factors in a group of subjects with definite diagnosis of Vit-D insufficiency or Vit-D sufficiency, and generation of a function which can be extended and applied to a population that the group of subjects are from and represent.

As used herein, the term â€œa population of interestâ€ means any population with common nature(s). For example, â€œa population of interestâ€ can be a population that lives in the same city, region, country or continent. Alternatively, â€œa population of interestâ€ can be a population that is within a certain age range. Also, â€œa population of interestâ€ can be a population that has same basic skin colour. As understood by a person skilled in the art, the more common features the population of interest shares, the more efficiency (e.g. sensitivity, specificity, accuracy etc.) the function will have. An appropriate selection of â€œa population of interestâ€ would be within the capability of a person skilled in the field of epidemiology. In some embodiments, the population of interest is an Asian population. In some embodiments, the population of interest is a Chinese population.

In some embodiments, in determining a Vit-D status of the method disclosed herein, a cohort of subjects of the population is statistically selected as being representative of the population, to create the function. Cohort study is commonly used in the field of epidemiology, and the qualification of a cohort would be appreciated by a person skilled in the art. In some embodiments, the subjects do not suffer from medical conditions that affect bone metabolism.

In some embodiments, determining a Vit-D status includes determining Vit-D insufficiency or Vit-D sufficiency in each subject in the cohort. The determination is based on serum 25(OH)Vit-D level assay. Serum 25(OH)Vit-D level is the best indicator of Vit-D status (19). In some embodiments, the assay can be performed using the gold standard of liquid chromatography tandem mass spectrometry (LCTMS) with details of method described previously (20, 21), as demonstrated in the Examples.

Obtaining a set of factors includes generating a series of factors associated with Vit-D status in a subject, including skin colourimetry measurements (constitutive skin melanin pigmentation measurement (Vf) and facultative skin melanin pigmentation measurement (Vh)) and other confounding factors, such as age, season, gender, body weight (BW), standing height (SH), physical activity level (PAL), daily dietary Vit-D intake (Dvd), daily dietary calcium intake (Dca) or any combination thereof. It shall be understood that transformed formats of these factors can also be used.

Constitutive skin melanin pigmentation measurement (Vf) can be measured using skin colourimetry on the instep area, the inner aspect or the sole of the foot, or the heel of individual subject. In some embodiments, Vf is measured on the non-dominant foot. In a particular embodiment, Vf is measured on the instep area of the non-dominant foot. Facultative skin melanin pigmentation measurement (Vh) can be measured using skin colourimetry on the dorsum of the hand, the fingers, the thumb or the wrist of individual subject. In some embodiments, Vh is measured on the non-dominant hand. In a particular embodiment, Vf is measured on the dorsum of the non-dominant hand. In some embodiments where an L*a*b* colour measurement system is used, Vf and Vh are in degrees.

In some embodiments, skin colourimetry is Individual Typology Angle (ITA) based on skin colourimetry, e.g. L*a*b* colour system. ITA is used to measure skin melanin pigmentation, and parameters for the sites to be measured. Skin colourimeters have been developed in the art, one example of which is CM-2300D Skin Colourimeter manufactured by Konica-Minolta which is exemplarily used in the Examples of the present application, and can generate the data of L and b from which the ITA is calculated. It shall be understood that skin colourimetry based on other mechanisms can also be used as long as skin colourimetry measurements can be generated for various body sites. Where other skin colourimetry systems are used, the Vf and Vh may be in units other than degrees.

Factors, such as age, gender, body weight (BW), standing height (SH), can be directly determined from a subject. Physical activity level (PAL) are well known to a person skilled in the art and can be readily measured. Daily dietary Vit-D intake (Dvd) and daily dietary calcium intake (Dca) can be estimated by a food frequency questionnaire. It shall be understood that the above factors can be expressed in different units, and as long as the unit of individual factor is consistent among the subjects, the function will work.

Among the factors, gender and season (i.e. the season when the skin colourimetry measurements are obtained) are two not expressed in values. Therefore, in order to create a function, these two factors can be differentially designated pre-set values.

In some embodiments, in season factors, summer is designated a value of â€œ1â€ and winter is designated a value of â€œ2â€ . It shall be understood that, in some embodiments where there are only two seasons (e.g. summer and winter) under consideration, the logistic regression model can be applied with the two seasons to be considered within the same logistic regression model. It shall be also understood that, in some embodiments where more than two seasons are considered, the logistic regression model can be evaluated with subgroup analysis, i.e. one at a time for each season. In this base, it still will be satisfactory with more than two seasons. Since for each season, a person skilled in the art can have a different logistic regression formula for predicting Vit-D status.

In some embodiments, in gender factors, male is designated a value of â€œ1â€ and female is designated a value of â€œ2â€ . It shall be understood that other designated pre-set values can also be used.

It shall be also understood that, besides Vf and Vh which are necessarily included in the factors, a person skilled in the art could make a selection among the other factors depending on actual needs and conditions.

Carrying out a logistic regression analysis of the method includes carrying out logistic regression analysis by using Vit-D status (â€œ0â€ or â€œ1â€ ) as the dependent variable, and the factors or the transformed formats of the factors as the independent variables, thereby obtaining a function with regression coefficients for each of the independent variables. Logistic regression model is commonly used in the field of statistics and well known to a person skilled in the art. General guidelines for logistic regression analysis can be easily found, e.g. http://en.wikipedia.org/wiki/Logistic_regression, or Y H Chan, â€œBiostatistics 202: Logistic regression analysis, Singapore Med J 2004 Vol 45(4): 149â€ which is incorporated herein by reference in its entirety.

In brief, logistic regression analyzes how a dependent variable can be predicted by a list of independent variables. In the embodiments of the present application, the dependent variable is a dichotomous variable, i.e. Vit-D status as 1=Vit-D insufficiency and 0=Vit-D sufficiency. For example, a logistic equation may have the following format:

$\log_{e} î¢ (\frac{Pred}{1 - Pred}) = Î± + {âˆ‘}_{1}^{k} î¢ž î¢ž {Î²}_{i} î¢ž x_{i}$

wherein Î± is constant term,

Î²_iis the regression coefficient for an independent variable x_i,

x_irepresent individual independent variables,

k represents the total number of independent variables,

Pred is the predicted probability used for determining the screening result of either Vit-D insufficiency or Vit-D sufficiency according to a pre-set cut-off threshold of Pred.

The function can be transformed to:

$\frac{Pred}{1 - Pred} = ï^{Î± + {âˆ‘}_{1}^{k} î¢ž î¢ž {Î²}_{i} î¢ž x_{i}}$ $Pred = \frac{1}{1 + ï^{- (Î± + {âˆ‘}_{1}^{k} î¢ž î¢ž {Î²}_{i} î¢ž x_{i})}}$

Logistic regression analysis can be performed by many computer programs, one example of which is SPSS software which is exemplarily used in the Examples of the present application.

Once a function is established, it can be used to screen Vit-D insufficiency by introducing the factors from a subject to be screened thereby generating a Pred, and comparing the calculated Pred with a pre-set cut-off threshold of Pred.

Determination of an appropriate pre-set cut-off threshold of Pred can be done according to the result from a logistic regression analysis. For example, from a logistic regression analysis, the sensitivity and specificity for each cut-off threshold can be generated. Thus, a person skilled in the art can consider the desired sensitivity and specificity (as high as possible) while making an appropriate balance between the sensitivity and specificity, and then determine pre-set cut-off threshold of Pred for individual solution. An exemplary determination of a pre-set cut-off threshold of Pred is demonstrated in the Examples. In some embodiments, the pre-set cut-off threshold of Pred is about 0.61.