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1. (WO2019042488) METHOD OF STANDARDIZED QUANTIFICATION OF COMPONENTS OF APOCRINE SWEAT
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Method of Standardized Quantification of Components of Apocrine Sweat

Field of Art

The present invention relates to a method of standardized quantification of components of apocrine sweat, enabling the comparison between their contents in different samples. A component of sweat refers to a chemical substance or a combination of substances, whose amount (quantity) is being determined. The use of this method enables the identification of markers of various diseases or conditions from apocrine sweat, as well as reproducibly diagnosing patients from apocrine sweat samples.

Background Art

Sweat is formed in apocrine or exocrine sweat glands localized in the epidermis. One of the primary functions of sweat is thermoregulation and control of body temperature by means of evaporation. Further, sweat is an important component of the skin protection mechanisms. Excretion of metabolites on the large area of the body surface enables the excretion of chemo-signalling molecules, e.g. androstadienone and other substances of pheromone character. Also, a number of products of metabolic processes, such as uric acid, are excreted through this process. The large area of the skin, occupied by exocrine and apocrine sweat glands, enables the excretion of a wide spectrum of small hydrophilic and larger lipophilic metabolites. These metabolites include amino acids, low-molecular amines, carboxylic acid, as well as proteins, antimicrobial peptides, xenobiotics, and drugs. Moreover, ethanol and explosives of the nitrate type are significantly excreted into sweat, if a person had contact with them prior to examination.

Although the biological matrix of the substances contained in sweat would theoretically be suited for diagnostics from the standpoint of analytical methodologies, its use in practice is substantially non-existent. Traditionally, the limited use of sweat samples for clinical diagnostics can be explained by a lack of studies concerning sweat composition, difficulties in maintaining the reproducibility due to variability in collected sample quantity, limited availability of suitable sweat collection methods, and, importantly, due to a great variability in the concentration of substances in sweat during the day as well as variations between the days in long-term observations extending over several days, and a huge inter-individual variability in the concentration of sweat components.

The distribution of the number and type of sweat glands, both exocrine and apocrine, differs according to body zone. Exocrine sweat glands prevail on the back and hand palms, whereas large concentrations of apocrine glands occur in the armpit and pubic area. Apocrine secretion of sweat glands has a lipophilic character, whereas the exocrine glands produce sweat mostly containing electrolytes and small organic molecules comprising under stabilized conditions 99 % of water.

There exists a need to find a standardized method of quantification of the components of sweat in a reproducible manner, thus enabling the comparison between different samples, regardless of sample collection method and inter-individual differences in sweat concentration. Most collection methods of apocrine sweat samples do not even allow the determination of collected sweat quantity, and there is no method for standardization of concentrations of sweat components. The provision of such a method would allow identification of relationships of the concentrations of components of sweat to various health conditions and development of diagnostic methods based on sweat samples.

Disclosure of the Invention

The present invention is based on the finding that in order to evaluate the contents of components in samples of apocrine sweat, logarithms of values expressing the amounts of the components in a sweat sample should be used. Furthermore, it is based on the finding that a suitable point of reference, to which values expressing the amounts of sweat sample components can be reliably standardized, is the arithmetic mean of logarithms of the values expressing the amounts of three components, which are further referred to as standardization markers:

These features enable standardization of the amounts or proportions of the components and a reproducible quantification which obviates the influence of sweat dilution or concentration, or of the variability of the methods of sample collection, or of the inter-individual variability. So far, no standardization marker or combination of markers was identified in sweat which could be employed as an internal standard. This invention provides the first reliable internal standard for reproducible determination of the quantitative amounts of components in apocrine sweat.

The need for logarithmic transformation of the measured values of the amounts of the components is based on the finding that the measured values have an approximately lognormal distribution, which is transformed by logarithmic operation to a desirable normal distribution.

The present invention thus provides a method of standardized quantification of components of apocrine sweat, said method comprising a step of obtaining at least one value xt expressing the amount of at least one component of apocrine sweat in a sample of apocrine sweat and obtaining values xj, ¾, ¾ expressing the amounts of the components palmitoleyl carnitine (SMI), hydroxyoctanoyl carnitine / pimeloyl carnitine (SM2), histidine (SM3) in the same sample; a step of subjecting the obtained at least one value xt expressing the amount of the at least one component of apocrine sweat to logarithmic transformation to obtain at least one loga(jCi); and a step of dividing the obtained values loga(jc;) by an arithmetic mean of logarithms of the values xj, ¾, ¾ expressing the amounts of the standardization markers palmitoleyl carnitine (SMI), hydroxyoctanoyl carnitine / pimeloyl carnitine (SM2), histidine (SM3) (i.e., (loga(xi) + loga(¾) + loga(¾))/3), thus obtaining the standardized values S,.

Generally, the logarithm base can be any positive number different from 1. The value 5, is independent of the value of the logarithm base a. However, the skilled person will appreciate that the same logarithm base a must be used for the logarithmic transformation of the values xt expressing the amount of the at least one component of apocrine sweat as well as for the logarithmic transformation of the values xj, ¾, ¾ expressing the amounts of the standardization markers palmitoleyl carnitine (SMI), hydroxyoctanoyl carnitine / pimeloyl carnitine (SM2), histidine (SM3).

The calculation of the standardized value Si can be expressed as follows:

s = logfl (*,-)

(loge ( xl ) + logfl (x2 ) + logfl ( x3 ) ) / 3

wherein:

Si is the standardized value expressing the amount of the component i in apocrine sweat, the standardized value is a value which can be compared between various samples;

Xi is the value expressing the amount of the component in the sample of apocrine sweat;

xi is the value expressing the amount of palmitoleyl carnitine (SMI) in the sample of apocrine sweat;

X2 is the value expressing the amount of hydroxyoctanoyl carnitine/pimeloyl carnitine (SM2) in the sample of apocrine sweat;

X3 is the value expressing the amount of histidine (SM3) in the sample of apocrine sweat; a is the logarithmic base.

The values xt expressing the amounts of the components of apocrine sweat in the sweat sample are obtained by measurement of the contents of the components of apocrine sweat in the sample by a suitable method. Suitable methods include methods of liquid sample analysis, such as by liquid chromatography. The values xt may be, for example, areas under chromatographic peaks, or values of molar, weight/mass or volume concentrations or proportions of the components. The values xj, ¾, ¾ expressing the amounts of the standardization markers in the same sample are typically obtained by the same method of measurement of the contents of the components of apocrine sweat as the component(s) to be standardized.

The components of apocrine sweat are substances occurring in apocrine sweat. In some embodiments, the components of apocrine sweat are hydrophilic substances occurring in apocrine sweat.

The sample of the apocrine sweat may be collected from the tested subject, in any suitable way, e.g. using a collecting device such as a sintered glass plate. The samples are usually extracted from the collecting device for the analysis. They are typically extracted by means of a solvent which may be an organic solvent, preferably a polar solvent (such as methanol or acetonitrile), and/or water.

This invention relates in particular to standardized quantification of human apocrine sweat components.

The method of the present invention can be used for standardization of markers in diagnostic methods from apocrine sweat samples, or in methods of identifying diagnostic markers for diagnostic methods from apocrine sweat.

Brief Description of Drawings

Fig. 1 shows the values r for the 142 studied samples of sweat. The physical quantity r is a ratio of the arithmetic mean of the logarithms of the values of the three standardization markers to the arithmetic mean of the logarithms of the values of the remaining 103 studied markers.

Fig. 2 shows the values of r for the studied samples of 73 persons suffering from breast cancer.

Fig. 3 shows the values of r for the studied samples of 50 persons not suffering from breast cancer.

Fig. 4 shows the values of r for the studied samples of 19 persons suffering from a benign breast tumor.

Figs. 5 to 9 show the continuous course of the distribution function of the normal distribution with the mean and the standard deviation corresponding to the mean and the standard deviation of the standardized values of the specific (arbitrarily selected) markers of all tested 142 persons.

Examples of carrying out the Invention

Example 1: Standardization significance of markers SMI, SM2, SM3

From a total of 142 women, wherein 73 women were diagnosed with breast cancer, 19 women were diagnosed with benign breast tumor, and 50 women were healthy (not suffering from breast cancer), samples of axilar apocrine sweat were collected. The samples were extracted by 80% aqueous methanol, centrifuged, and the supernatant was used for the analysis.

Liquid chromatography was used for separation of sweat components, specifically the system UltiMate 3000 RS (Dionex, Sunnyvale, CA, USA). The separation was performed on the Luna NH2 3.0 μιη, 2 x 100 mm column, protected by a protective column, 4 x 2 mm ID,

made of an identical material (Phenomenex, Torrance, USA), at 35 °C in HILIC mode. Mobile phases A (20 mM ammonium acetate, pH 9.75) and B (acetonitrile) were used at a flow rate of 0.3 mL/min. Total time of the analysis was 17 minutes, using a gradient elution: 0-7 min: 95%→ 10% B; 7-13 min: 10% B; 13- 14 min: 10%→ 95% B; 14-17 min: 95% B. Mass spectrometer with triple quadrupole QTRAP 5500 (SCIEX, Framingham, MA, USA) with electrospray ionization and positive/negative polarity switching was used for the detection. In order to increase sensitivity, the measured components were scanned in the multiple reaction monitoring (MRM) mode with an extended delay (MRM detection time: 60 s; scanning time: 0,8 s; settling time: 50 ms). Both quadrupoles were set to unit resolution. Parameters of the ion source and of the gas were: ion electrospray voltage: + 4500 V; gas countercurrent: 30 psi; gas in the ion sources: 40 psi and source temperature: 400 °C. High-purity nitrogen was used as the collision gas (pressure set to "medium"), as the gas countercurrent, and as the gas in the ion sources. Declustering potentials, input potentials, collision energies and output potentials of the collision cell were optimized for each metabolite, using standards. The apparatus was controlled by the software Analyst 1.6.2 (SCIEX, Framingham, MA, USA).

Values x (areas under curve in the chromatogram) were measured for 106 preliminarly tested markers, including the 3 markers for which the standardization character was proven during the tests.

The standardization character of the three markers of the invention is demonstrated by the fact that the ratio r of the arithmetic mean of the logarithms of the values of said three standardization markers to the arithmetic mean of the logarithms of the values of the 103 remaining tested markers is nearly constant in all 142 tested samples, i.e., the expression


has nearly the same value (about 1.2) for all samples (k = I , ... , 142). This was not observed for any other marker or combination of a small number of markers.

The extent of relative standard deviations from the average value of r* may be expressed by a variation coefficient, i.e., a ratio of the standard deviation of the whole set of all values r* to the arithmetic mean of these values.

The figures show the values of r for apocrine sweat samples:

Fig. 1 shows the values of r for all 142 sweat samples.

Fig. 2 shows the values of r for 73 sweat samples obtained from persons suffering from breast cancer.

Fig. 3 shows the values of r for 50 sweat samples obtained from persons not suffering from breast cancer.

Fig. 4 shows the values of r for 19 sweat samples obtained from persons suffering from benign breast tumor.

The results shown in Figures 1 to 4 are summarized in the following table :


These results show that a possible but impractical method of standardization by dividing a value of the observed component by an arithmetic mean of the values representing the amounts for a large number of the sweat components can be much more practically replaced by the standardization method of the invention, using an arithmetic mean of the values representing the amounts of only three markers. The relative deviation of the results obtained by these two methods is characterized by a variation coefficient of only about 2 %.

The benefits of the method of standardization according to the invention are further demonstrated by the fact that the standardized markers possess a distribution close to the normal one in the whole set of 142 samples. In Figs. 5 to 9, the continuous curves express the real course of the distribution function of normal distribution with the mean and standard deviation corresponding to the mean and standard deviation of the standardized values of specific (arbitrarily selected) markers of all 142 persons tested. These individual values are presented in the graph as points. A good agreement with the normal distribution in most markers under study is remarkable also due to the fact that they are obtained from samples of biological material.

Example 2: Example of use of the method of standardized quantification in a method of identification of diagnostic markers and/or in a method of diagnosis

Samples treated in the same way as in the previous example were used for identification of diagnostic markers of breast carcinoma.

First, markers were standardized using the method of the present invention, using the equation:


Afterwards, weight of markers of an evaluated person is determined as a logarithm of the ratio of probability of the person belonging to the group of persons suffering from breast cancer to probability of the person belonging to the group of persons not suffering from breast cancer. During the tests and evaluations it was determined that the best results are achieved by utilization of statistical dependencies between pairs of markers, in particular for 7 marker pairs (Mx , MY) shown in Table la, lb.

The calculation can be expressed as follows (for more details, see the co-pending application „Method of Breast Cancer Diagnostics from a Sample of Apocrine Sweat" filed by the same applicant on the same day):


acx +bc,SresC) fK<x) = f<y \ aKx +bK,SresK) (¾


wherein fc is the normal distribution probability density function of standardized values of the marker for persons suffering from breast cancer, /κ is the normal distribution probability density function of standardized values of the marker for persons not suffering from breast cancer, X is the standardized value of the marker Mx of the evaluated person, Y is the standardized value of the marker Μγ of the evaluated person, W is the weight of marker of the evaluated person.

For persons suffering from breast cancer Yc = acX +bc sresC , (4)

For persons not suffering from breast cancer YK = aKX +bK sresK (5) wherein sres are residual deviations.

The resulting evaluation of the person (classification score S) is defined as sum of weights for all pairs of markers:

S =∑W, (6)

=1

The thus defined weight is a positive number for persons with a higher probability of belonging to the group of persons suffering from breast cancer and a negative number for persons with a higher probability of belonging to the group of persons not suffering from breast cancer.

The density values were limited by the value 0.05 as the minimum value. Lower value were replaced by the limit value.

Parameters of linear regressions a, b, sres shown in the table were obtained by censoring method applied to standardized values X ,Y of these markers. Data of 10 persons with the most advanced state of breast cancer and 27 persons not suffering from breast cancer were used for the computation. A higher number of healthy persons was used because of high variability of their data.

Table la


Table lb

Designation of

Chemical identity

marker

Ml carnitine

M3 decenoyl carnitine

M6 glutamine

M7 proline

M8 betaine

M9 5-oxoproline

M14 lactic acid

glutaconic acid / ketoleucine / mevalonolacton / 3-methyl-2- M15

oxopentanoic acid

M17 adenosine

M19 4-hydroxybenzaldehyde

M20 hydroxyoctanoyl carnitine / pimeloylcarnitine

Examples of evaluation of individual samples are shown in tables 2 and 3:

Table 2


Negative value of the score means the classification "not suffering from breast cancer".

For evaluation of the data of 70 persons suffering from breast cancer and 53 persons not suffering from breast cancer, sensitivity of 97 % (2 cases of unrecognized cancer) and specificity of 72 % (15 persons wrongly evaluated as suffering from breast cancer) was achieved.