A technique for determining functionality of a physiological membrane

The present technique is related to determining functionality of a physiological membrane.

Biological membranes or bio-membranes, also referred to a physiological membrane, found in living organisms, such as animals and humans, are responsible for performing various functions in the body of the organism. A very important functionality of certain physiological membranes is to carry out filtration of fluids in the body of the organism, for example Glomerular filtration of blood performed in kidney of animals. When the filtration functionality of kidney is affected it is symptomatic of some pathological condition in the animals such as CKD (chronic kidney disease) in humans. CKD results from loss of kidney function over an extended period of time. Reduction in renal glomerular filtration rate (GFR) is indicative of and helps in determining severity of CKD. In people affected by CKD, the glomeruli of the kidney lose their ability to filter out substances, including several toxins, from the blood plasma over their membranes towards the glomerular filtrate i.e. the urine. Due to lack of proper filtration functionality of the kidney several pathological conditions may develop for example build up of toxins in the blood of the patient. Main causes of the disease are hypertension, glomerulonephritis and diabetes mellitus. The disease if untreated progresses from a mild form towards more severe forms, finally resulting in end- stage renal disease. Thus, assessing functionality of a physiological membrane is very useful for various purposes. The knowledge of functionality of physiological membranes such as the renal glomerular filtration system, the alveolar tissue of the lungs, and so on and so forth is important for determination of different pathological conditions as well as for therapy monitoring and guidance.

Hence reliable and cost-effective techniques are needed to assess the changes in functionality of the physiological membranes such as changes in kidney filtration functionality. Monitoring functionality of the physiological membranes may be helpful to detect malfunctioning or loss of functionality of such physiological membranes at an early stage, which may in turn trigger delivery of early treatment and life style changes. Advantageously, the technique of monitoring or determining the functionality of the physiological membrane should be non-invasive, i.e. they would require no blood draw, in order to promote wide application and to include test subjects who are opposed or unwilling to invasive techniques .

Assessment of GFR has been conventionally carried out in various ways. One conventional way of assessing loss of kidney function is by using retained urea and creatinine in urine and blood samples. However, this method requires drawing of blood sample from the test subject by invasive methods, for example by using a syringe needle to draw blood sample, and thus is avoided by people having a fear of or unwillingness to invasive methods.

A non-invasive technique conventionally known is to use recognition of biomarkers present in exhaled breath of a test subject. However, this technique needs involvement of highly sophisticated methods and instruments and expertise of trained operators .

Thus, the object of the present technique is to provide a simple technique for determining functionality of a

physiological membrane of a test subject. The technique is desired to be noninvasive. The above objects are achieved by a method for determining functionality of a physiological membrane according to claim 1 and by a device for determining functionality of a

physiological membrane according to claim 10 of the present technique. Advantageous embodiments of the present technique are provided in dependent claims. Features of claim 1 may be combined with features of dependent claims, and features of dependent claims can be combined together. Similarly, features of claim 11 may be combined with features of dependent claims, and features of dependent claims can be combined together.

According to an aspect of the present technique, a method for determining functionality of a physiological membrane of a test subject is presented. In the method a concentration of a group of selected Volatile Organic Compound (VOC) in a test medium is determined. The test medium is a filtrate of the physiological membrane. In the method, and independent of the previous step, a concentration of the group of selected VOC in a reference medium is determined. The reference medium is not a filtrate of the physiological membrane. Subsequently, a test quotient is calculated from the concentration of the group of selected VOC in the test medium and the

concentration of the group of selected VOC in the reference medium. Finally, the test quotient is compared to a reference range. The reference range includes a range of quotients corresponding to the group of selected VOC and representing a known state of functionality of the physiological membrane. This provides a simple non-invasive technique to determine the functionality of the physiological membrane.

In the method, in comparing the test quotient to the

reference range, the known state of functionality is one of a physiologically normally functioning state and a

physiologically abnormally functioning state. Thus if the test quotient falls within or overlaps with the reference range of quotients representing the physiologically normally functioning state, then it may be concluded that the physiological membrane is functioning as desired in a healthy test subject, however, if the test quotient falls within or overlaps with the reference range of quotients representing the physiologically abnormally functioning state, then it may be concluded that the physiological membrane is not

functioning as desired in a healthy test subject. Moreover, by the comparison of the test quotient with the reference range the severity of abnormality may also be determined. In another embodiment of the method, the test medium is a urine sample obtained from the test subject. Thus, the method is useful to determine a normal or abnormal glomerular filtration rate. In another embodiment of the method, the concentration of the group of selected VOC in the test medium is determined by determining the concentration of the group of selected VOC in a head space of the urine sample. Since the VOC is volatile in nature with passage of time the VOC escapes from the urine into the head space and collects in the headspace when stored in a container. The head space of the urine thus represents the composition of VOC in the original urine sample.

In another embodiment of the method, the reference medium is either an exhaled breath sample obtained from the test subject or an air over skin sample obtained from the test subject. Thus the method is simplified by use of the

reference medium which are easy to collect and do not need an invasive method for collection.

In another embodiment of the method, the group of selected VOC comprises only one type of VOC. Thus the method is simplified because only one type of VOC is determined and this needs a use of simple sensor or analytical instrument or simple method of determining.

In another embodiment of the method, the group of selected VOC comprises a plurality of different types of VOC. By using more than one type of VOC, the range of the method is improved and also the sensitivity of the method is improved as the method in this embodiment takes into consideration more than one type of VOC.

In another embodiment of the method, the type of VOC in the group of selected VOC is a commonly occurring VOC for the filtrate of the physiological membrane. The commonly

occurring VOC is not a biomarker or is not representative of a biomarker for a specific pathological condition of the physiological membrane. Thus, there is no need of detecting special types of VOCs that are present only in diseased individuals and thus the method is not dependent on special type of VOCs thereby increasing the applicability of the method.

In another embodiment of the method, the concentration of the group of selected VOC in the test medium and the

concentration of the group of selected VOC in the reference medium are determined by using one of Gas Chromatography (GC) , Mass spectrometry (MS) , Gas Chromatography-Mass

Spectrometry (GC-MS) , Gas Chromatography-tandem Mass

Spectrometry (GC-MS/MS) , Ion-mobility spectrometry (IMS), Solid-phase microextraction Gas Chromatography-Mass

Spectrometry (SPME-GC-MS) , and a combination thereof. These present simple and reliable techniques to implement the method of the present technique.

According to another aspect to the present technique, a device for determining functionality of a physiological membrane of a test subject is presented. The device includes a first module, a second module, a test quotient calculating module, and a comparing module. Optionally, the device includes a memory module. The first module provides a first value representing a concentration of a group of selected Volatile Organic Compound (VOC) in a test medium. The test medium is a filtrate of the physiological membrane. The second module provides a second value representing a concentration of the group of selected VOC in a reference medium. The reference medium is not a filtrate of the

physiological membrane. The test quotient calculating module receives the first value from the first module and the second value from the second module. Subsequently, the test quotient calculating module calculates a test quotient from the first value and the second value. The comparing module compares the test quotient to a reference range. The reference range includes a range of quotients corresponding to the group of selected VOC and representing a known state of functionality of the physiological membrane.

In an embodiment of the device, the first module receives the first value from an analytical device and the second module receives the second value from the analytical device. Thus the analytical device analyses the test medium and the reference medium and provides the first and the second values to the device for determining the functionality of the physiological membrane. Thus analysis of test medium and the reference medium may be done remotely from the device for determining the functionality of the physiological membrane, and only the results of the analysis may be provided to the device of the present technique. In the embodiment of the device including the memory module, the memory module stores the reference range. In this

embodiment, the comparing module obtains the reference range from the memory module. Thus the requirement to provide the reference range from an external source at every usage of the device is obviated.

In another embodiment of the device the group of selected VOC includes only one type of VOC. Thus the device is simple and requires less computational power.

In another embodiment of the device the group of selected VOC includes a plurality of different types of VOC. By using more than one type of VOC, the range of the device is improved and also the sensitivity of the device is improved as the device in this embodiment takes into consideration more than one type of VOC. In another embodiment of the device, the type of VOC in the group of selected VOC is a commonly occurring VOC for the filtrate of the physiological membrane and not representing a biomarker for a specific pathological condition of the physiological membrane. Thus, there is no need of detecting special types of VOCs that are present only in diseased individuals and thus the device is not dependent on special type of VOCs thereby increasing the applicability of the device . The present technique is further described hereinafter with reference to illustrated embodiments shown in the

accompanying drawing, in which:

FIG 1 illustrates a flow chart representing a method for determining functionality of a physiological membrane of a test subject, and

FIG 2 schematically illustrates a device for determining functionality of the physiological membrane of the test subject, in accordance with aspects of the present technique.

Hereinafter, above-mentioned and other features of the present technique are described in details. Various

embodiments are described with reference to the drawing, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purpose of explanation, numerous specific details are set forth in order to provide a thorough understanding of one or more embodiments. It may be noted that the illustrated embodiments are intended to explain, and not to limit the invention. It may be evident that such embodiments may be practiced without these specific details. The idea of the technique is to use one or more Volatile Organic Compound (VOC) naturally generated and/or excreted from body of an organism. The rate of excretion for a given VOC is dependent primarily on the following:

a) the concentration difference of the given VOC in the bloodstream and on the other side of a physiological

membrane, for example when the physiological membrane is glomerular capillaries, the difference in concentration of VOC in blood stream and concentration of VOC in glomerular filtrate,

b) the permeability of the physiological membrane for

example, lung tissue, skin, kidney tissue (predominantly the glomeruli) , and

c) the physicochemical properties of the given VOC (e.g.

hydrophobicity, length of side chains, etc.), respectively.

The concentration of the VOC on one side of the physiological membrane i.e. say the bloodstream side is in most cases dependent on age, gender, weight, size, body mass index, and distribution of fat and muscle tissue, and could be

determined for a healthy individual with properly functioning physiological membrane and be recorded in a reference value table. However, the concentration of the VOC on the other side of the physiological membrane results primarily from result of filtration performed by the physiological membrane, and thus if the filtration performed by the physiological membrane is excluded the concentration of the VOC on the other side of the physiological membrane is negligible.

If now a selected VOC is excreted or filtered over one membrane (e.g. the lung) at a certain rate, and at another membrane (e.g. the kidney glomeruli) at another rate, a quotient can be built by measuring the concentration of the substance in the first medium (exhaled air in this case) and from the other medium (from a headspace or directly from urine in this case) from the same donor or same test subject. When due to changes in the permeability of one of these membranes the diffusion of the selected VOC is disturbed, then this can be detected by a shift of the quotient of the concentration of the VOCs in the two media. Thus a shift in the quotient of the concentrations of VOCs on the two sides of the physiological membrane is directly representative of a change in the permeability of the physiological membrane. A change in the permeability of the physiological membranes can be caused by pathologic processes, e.g. by a change in GFR due to kidney disease which affect functionality of the physiological membrane. Hence the functionality of the physiological membrane, for example GFR of an individual i.e. the test subject can be determined by measuring the

distribution of the selected VOC in a test sample, say the urine sample, say directly from urine or from the headspace of the urine, from the individual and from a reference sample, say the exhaled breath or from air over skin. The same principle can be applied to measure the permeability of any physiological membrane. Referring to FIG 1, a flow chart representing a method 1000 for determining functionality of a physiological membrane of a test subject is presented. In the method 1000, a

concentration of a group of selected Volatile Organic

Compound (VOC) in a test medium is determined in a step 100. The test medium is a filtrate of the physiological membrane. The test subject may be an animal or a human being. In an embodiment the test medium is glomerular filtrate i.e. urine and thus the physiological membrane is glomerular capillaries or simply glomeruli. The concentration of the group of selected VOC may be measured directly in the urine sample, or in a headspace of the urine sample.

Furthermore, in the method 1000, and independent of the step 100, a concentration of the group of selected VOC in a reference medium is determined in a step 200. The reference medium is not a filtrate of the physiological membrane. In an embodiment the reference medium is an exhaled breath from the test subject or a sample of air collected from over a skin of the test subject.

The group of selected VOC may be one type of VOC or may be a plurality of VOC. In an embodiment of the method 1000, the type of VOC in the group of selected VOC is a commonly occurring VOC for the filtrate of the physiological membrane. The commonly occurring VOC is not a biomarker or is not representative of a biomarker for a specific pathological condition of the physiological membrane. Thus, the type of

VOC is one or more VOC that are present in normal conditions as well in pathological conditions in the filtrate of the physiological membrane of a individual suffering or not suffering from a pathological condition associated with the physiological membrane. To explain further, the type of VOC is one that is not a biomarker of CKD or other pathological conditions of the physiological membrane.

The type of VOC may be one of the most represented chemical classes generally found in the filtrate of the physiological membrane. For example when the functionality of the

physiological membrane being determined is the glomerular filtration rate then the VOC may be a ketone for example acetone, 2-butanone, 3-methyl-2-butanone, 3-methyl-2- pentanone, 2-pentanone, so on and so forth, and/or an

aldehyde for example propanal, 2-methylpropanal, 2-methyl- butanal, and so on and so forth, and/or a sulfur containing compound for example methanethiol , dimethyl sulfide (DMS) , etc .

The concentration of the group of selected VOC in the test medium and the concentration of the group of selected VOC in the reference medium may include concentrations of different types of VOCs when the group of the selected VOC includes more than one type of VOC.

The concentration of the group of selected VOC in the test medium and the concentration of the group of selected VOC in the reference medium are determined, in the step 100 and step 200, respectively, by using any conventionally known

analytical techniques for example Gas Chromatography (GC) , Mass spectrometry (MS) , Gas Chromatography-Mass Spectrometry (GC-MS) , Gas Chromatography-tandem Mass Spectrometry (GC- MS/MS), Ion-mobility spectrometry (IMS), Solid-phase

microextraction Gas Chromatography-Mass Spectrometry (SPME- GC-MS) , and a combination thereof. The principle of use and application of these techniques are well known in art of analytical chemistry and thus not described herein in details for sake of brevity.

Subsequently, in the method 1000, a test quotient is

calculated in a step 300 from the concentration of the group of selected VOC in the test medium and the concentration of the group of selected VOC in the reference medium. The test quotient may simply be a mathematical quotient of the

concentration of the group of selected VOC in the test medium and the concentration of the group of selected VOC in the reference medium. In the method 1000, finally, the test quotient is compared in a step 400 to a reference range. The reference range includes a range of quotients corresponding to the group of selected VOC and representing a known state of functionality of the physiological membrane.

In an embodiment of the present technique, in the method 1000, the functionality of the physiological membrane is a state of filtration of the physiological membrane, for example, a normal state or a healthy state of filtration of the physiological membrane meaning thereby that function of filtration as performed by the physiological membrane is healthy or representative of normal or common function as seen in an healthy subject. Another example of the state of filtration of the physiological membrane may be an abnormal state or an unhealthy state of filtration of the

physiological membrane meaning thereby that function of filtration as performed by the physiological membrane is not healthy or representative of disease ridden or malfunctioning physiological membrane.

As used herein the term 'reference range' refers to a range of quotients for a defined set of individuals of a relevant demographic group to which test sample results i.e. the test quotient may be compared. The size and characteristics of the set of individuals and the relevant demographic group may vary from one application of the method 1000 to another. In an embodiment, the reference ranges are based on observed results for a large number of individuals for example, collected in clinical trials. The reference range may include range limits, generally a range of standard deviations from the average result. The reference range may include quotients calculated from a first medium that is a filtrate of the physiological membrane similar to the test medium and the second medium that is similar to the reference medium, wherein the quotients represent a range of values that can be attributed to a normally or clinically healthy functioning physiological membrane comparable to the physiological membrane being probed. In such cases if the test quotient is within the reference range the functionality of the

physiological membrane is concluded to be normal. In another embodiment, the reference range may include quotients calculated from a first medium that is a filtrate of the physiological membrane similar to the test medium and the second medium that is similar to the reference medium, wherein the quotients represent a range of values that can be attributed to an abnormally or physiologically malfunctioning physiological membrane comparable to the physiological membrane being probed. In such cases if the test quotient is within the reference range the functionality of the

physiological membrane is concluded to be abnormal or

diseased.

In yet another embodiment, the reference range may include at least two sub-ranges. At least one of the two sub-ranges, say a first sub-range, includes quotients calculated from a first medium that is a filtrate of the physiological membrane similar to the test medium and the second medium that is similar to the reference medium, wherein the quotients of the first sub-range represent a range of values that can be attributed to a normally or clinically healthy functioning physiological membrane comparable to the physiological membrane being probed. At least one of the two sub-ranges, say a second sub-range, includes quotients calculated from a first medium that is a filtrate of the physiological membrane similar to the test medium and a second medium that is similar to the reference medium, wherein the quotients of the second sub-range represent a range of values that can be attributed to an abnormally or physiologically malfunctioning physiological membrane comparable to the physiological membrane being probed. In this embodiment, if the test quotient is within the first sub-range the functionality of the physiological membrane is concluded to be normal, however, if the test quotient is within the second sub-range the functionality of the physiological membrane is concluded to be abnormal or diseased.

Thus in the method 1000, in comparing the test quotient to the reference range in the step 400, the known state of functionality is either a physiologically normally

functioning state and/or a physiologically abnormally

functioning state. Thus if the test quotient falls within or overlaps with the reference range of quotients representing the physiologically normally functioning state, then it may be concluded that the physiological membrane is functioning as desired in a healthy test subject, however, if the test quotient falls within or overlaps with the reference range of quotients representing the physiologically abnormally

functioning state, then it may be concluded that the

physiological membrane is not functioning as desired in a healthy test subject. Referring to FIG 2, a device 1 for determining functionality of a physiological membrane of a test subject is presented. The physiological membrane and the test subject are same as the physiological membrane described in relation to FIG 1. The device 1 includes a first module 10, a second module 20, a test quotient calculating module 30, and a comparing module 40. In an embodiment of the device 1, the device 1 includes a memory module 50. The first module 10 provides a first value representing a concentration of a group of selected Volatile Organic

Compound (VOC) in a test medium. The test medium is a

filtrate of the physiological membrane and is same as the test medium described in reference to FIG 1. The second module 20 provides a second value representing a

concentration of the group of selected VOC in a reference medium. The reference medium is not a filtrate of the

physiological membrane and is same as the test medium

described in reference to FIG 1. The VOC in the group of selected VOC may be of one or more type as described in relation to FIG 1. The test quotient calculating module 30 receives the first value from the first module 10 and the second value from the second module 20. Subsequently, the test quotient calculating module 30 calculates a test

quotient from the first value and the second value. The comparing module 40 compares the test quotient to a reference range. The reference range includes a range of quotients corresponding to the group of selected VOC and representing a known state of functionality of the physiological membrane. The test quotient and the reference range are same as the test quotient and the reference range described in reference to FIG 1.

In an embodiment of the device 1, the first module 10 receives the first value from an analytical device 90 and the second module 20 receives the second value from the

analytical device 90. Thus the analytical device 90 analyses the test medium and the reference medium and provides the first and the second value to the device 1 for determining the functionality of the physiological membrane. The

analytical device 90 is a device using any conventionally known analytical techniques for example Gas Chromatography (GC) , Mass spectrometry (MS) , Gas Chromatography-Mass

Spectrometry (GC-MS) , Gas Chromatography-tandem Mass

Spectrometry (GC-MS/MS) , Ion-mobility spectrometry (IMS), Solid-phase microextraction Gas Chromatography-Mass

Spectrometry (SPME-GC-MS) , and a combination thereof. The principle of use and application of such analytical devices

90 are well known in art of analytical chemistry and thus not described herein in details for sake of brevity.

The first module 10 and the second module 20 may receive from the analytical device 90 values that are the concentration of the group of selected VOC in the test medium and the

reference medium, respectively or may receive from the analytical device 90 other values that are then used by the first module 10 and the second module 20 to calculate the concentrations of the group of selected VOC in the test medium and the reference medium. The first and the second values may be received by the first module 10 and the second module 20, directly from the analytical device 90 or may be received through an intermediate link for example by manual input by an operator who receives the first and the second values and then feeds them into the first module 10 and the second module 20, respectively. Thus analysis of test medium and the reference medium may be done remotely from the device 1, and only the results of the analysis may be provided to the device 1 of the present technique.

In the embodiment of the device 1 that includes the memory module 50, the reference range is stored in the memory module 50. In this embodiment of the device 1, the comparing module 40 obtains the reference range from the memory module 50. Alternatively, in the device 1 with or without the memory module 50, the reference range may be provided from an external source at every usage of the device 1. While the present technique has been described in detail with reference to certain embodiments, it should be appreciated that the present technique is not limited to those precise embodiments. Rather, in view of the present disclosure which describes exemplary modes for practicing the invention, many modifications and variations would present themselves, to those skilled in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.