METHOD FOR DETECTION OF A PHYSIOLOGICAL ABNORMALITY

Abstract: The present invention relates to an assay method for detecting a physiological abnormality in a human or animal. The invention is particularly described in relation to human hypertension (high blood pressure) in an adult study group and as a predictive marker for hypertension in an adolescent study group. In a particular embodiment there is described the use of human cheek epithelial cells to measure sodium ion (Na<+>) transport using radiolabelled <22>Na<+>. Also disclosed is the use of cheek cell Na<+> transport activity as a predictive marker for hypertension.

Description: Method for Detection of a Physiological Abnormality Technical Field The present invention relates to an assay method for detecting a physiological abnormality such as hypertension in a human or animal, and to assays and kits for use in the method. Hypertension is considered a major risk factor for heart disease. Untreated hypertension generally leads to irreversible damage to the heart and vasculature and undoubtedly shortens life. Cardiovascular problems associated with hypertension have decreased greatly during the last decade due to a combination of greater community awareness of the problem, early diagnosis by physical examination, and efficacious treatment of the condition once detected via the wide spectrum of antihypertensive medications currently on the market. In some cases hypertension can also be successfully contained by a combination of diet and lifestyle changes but the condition must be recognised and treated early if later deleterious effects are to be avoided. Nevertheless, the fact remains that hypertension is the commonest of the diseases affecting the heart and blood vessels. Studies from the National Heart Foundation's Risk Factor Prevalence Study (1980) indicate that one in five men and one in six women in Australia have high blood pressure. By 50 years of age, 31 % of men and 28% of women in Australia are hypertensive and by age 60 years this has increased to 47% and 39% respectively. At present only about half of these persons are aware of their condition and only about one third are being successfully treated. Most people suffering from the commonest variety of high blood pressure (essential hypertension which is of unknown aetiology), develop the disease in their thirties. However, during its early stages, it rarely produces symptoms. Unless a physical examination reveals that the blood pressure is high, a person may have the disease for many years without knowing. In general, they may be destined to develop hypertension at some time in the future due to their genetic predisposition. In terms of its inheritance, essential hypertension is understood to develop from a polygenic predisposition whose expression is enhanced by environmental mechanisms such as diet and whose outcome is fixed by inevitable structural changes due to the elevated blood pressure itself (Williams et al., 1990a,b). The development of reliable detection techniques for the determination of physiological abnormalities such as the predisposition to hypertension is clearly of benefit in allowing early treatment. To date there is no satisfactory method to identify pre-hypertensive children, except, perhaps, for the long-term procedure of blood pressure tracking. Standardised measurements of a child's blood pressure every two or three years during growth may show a consistently elevated pressure compared with other children of the same age, sex and body mass index and this will indicate a likelihood that the child will become a hypertensive adult: Blood pressure generally rises steadily with age and the blood pressure defined as mild hypertensive (95mmHg diastolic and/or 150mmHg systolic pressure) are rarely encountered in children. Nevertheless, the tendency to track in an upper or lower percentile rank is reasonably strong and is a good predictor of the adult ranking (Kneisley et al., 1990). Clearly the development of a simple diagnostic test which is capable of detecting both the pre-hypertensive individual and those adults exhibiting labile blood pressure readings which make a definite diagnosis difficult, would herald a major breakthrough in early and definitive diagnosis and would allow for earlier treatment. Such a diagnostic test would be all the more acceptable if it had wide application such as in the testing of children of hypertensive parents. Furthermore, if the test is essentially non-invasive in its nature, it need not be confined to the above situations, but could be more wide reaching and applied to other "at-risk" groups in the community. Background Art The most desirable form of test or predictive marker would be one which is indicative of some basic biochemical abnormality or defect in the hypertensive individual or in those individuals who would later develop hypertension, and which is likely to be related to the genetic predisposition of an individual to develop hypertension. Certainly no form of biochemical assay presently exists to detect those who may be predisposed to develop hypertension. Furthermore no biochemical test or assay has been developed which correlates unequivocally with all established hypertensive individuals. Existing biochemical assays appear to identify only certain subsets of hypertensives, i.e. only a certain proportion of hypertensive individuals display biochemical activities which are significantly different from the values determined for a comparable set of normotensive individuals (Garay, 1987). In this regard, the frequencies of Na + transport abnormalities in the red blood cells of Caucasian hypertensive subjects and the resulting change in the concentration of Na + in the red blood cell have revealed- abnormalities in four major Na + transport pathways. However, as stated above, the extent to which these individual pathways are abnormal in hypertension is certainly not uniform, nor is the ability of these Na + transport abnormalities to discriminate between hypertensive and normotensive individuals, definitive. The maximal rate of red blood cell Na + -Li + countertransport activity has been reported to be increased in only 20% to 50% of subjects in a hypertensive study group while that of the (Na + + K + ) pump was increased in only 5% to 15% of the hypertensive group. An increased uptake of Na + into the red blood cell via a passive "leak" pathway was reported to be present in only 10% to 30% of hypertensive individuals (Garay, 1987). The above are clear examples of the failure of existing tests to clearly and totally discriminate between hypertensive and normotensive individuals. Furthermore, they do not address identification of the pre-hypertensive individual. Existing tests developed by others rely on the measurement of Na + ,Li + or H + ion fluxes (whether singularly or in various combinations) across red blood cell, leucocyte or platelet cell membranes. Measurement of these ion fluxes have been done by direct means or indirectly by following cellular pH changes or swelling of a particular cell type. These tests have only been used in respect of adult subjects with established essential hypertension who have been compared with an appropriate control group of normotensive subjects who are matched for age, sex and body mass index. A complete list of these twenty or more studies are summarised in Appendix 6 and the full reference to each work is cited in the reference section. It must be pointed out that for some of these tests up to 120ml of blood must be sampled in order to carry out such assays. Use of these tests in the case of pre-hypertensive subjects or with children has to our knowledge not been reported. The use of cheek cells in studies on existing hypertensives has also to our knowledge not been exploited by others. Disclosure of the Invention It is an object of this invention to provide an alternative and more sensitive method for the determination of a physiological disorder such as essential hypertension in man. We have found that the presence of the above disorder may be reflected in the activity of a biochemical marker present in epithelial cells, the marker being the flow of ions, particularly Na + and H + , across the membrane of the epithelial cell. This marker may be used to discriminate between subjects having the disorder and those who have not, such as has been done in an adult study group. Furthermore this marker may be used to discriminate between subjects who have yet to develop this disorder but are considered to be at the greatest risk of later developing hypertension due to the combination of blood pressure tracking characteristics and family history of hypertension, as has been done in an adolescent study group. Accordingly, in a first aspect, the present invention provides a method for the determination of a physiological abnormality in a human or animal subject, which method includes determining ion flux across the membrane of epithelial cells taken from said subject. The epithelial cell may be a skin dermal epithelial cell, a bladder epithelial cell (eg that contained in urine), a nasal epithelial cell and the like. A particularly preferred cell is the cheek epithelial (buccal mucosal) cell. In another aspect, the present invention provides an assay for use in the determination method of the invention said assay comprising determining the flux of a first ion across the membrane of epithelial cells under an imposed concentration gradient of a second ion. The first and second ions may be cations. The first ion may be the sodium ion (Na + ), the second ion (H + ) and the epithelial cell such as the cheek epithelial (buccal mucosal) cell. The invention will now be more particularly described in reference to the determination of hypertension and/or pre-disposition towards hypertension in humans. The biochemical marker assayed for the determination of human hypertension (or pre-hypertension) is based on the rate of proton-dependent sodium ion (Na + ) uptake that occurs in epithelial cells such as cheek epithelial cells when measured as a function of the imposed proton gradient. It is likely, although not proven, that such activity is due in part to the Na + /H + antiporter or exchanger which has been argued by others to be a component activity of the Na + /Li + exchanger which is frequently measured in red blood cells and other cells derived from the blood in relation to hypertension (see Appendix 6). Thus, in yet a further aspect, the present invention provides a method for the determination of hypertension and/or pre-disposition towards hypertension in a human subject, said method comprising the steps of: establishing a proton (H + ) concentration gradient across the membrane of epithelial cells taken from said subject, the proton concentration being greater inside said cells than outside said cells; and determining the proton-dependent uptake of sodium ions (Na + ) by said cells when said cells are exposed to medium containing sodium ion (Na + ). Preferably the cells are exposed to a medium containing a final concentration in the range 50 micromole to 150 millimole per litre Na + . More preferably the cells are exposed to a medium containing a final concentration of about 1 millimole per litre Na + . The proton-dependent Na + transport may be determined directly by, for example, determining the uptake of radioactively labelled Na + (i.e. 22 Na + ). It may also be determined indirectly by using a probe, such as a fluorescent probe specific for Na + . Changes in proton-dependent Na + uptake could also be determined by measuring proton (H + ) movements across the cell membrane of an epithelial cell such as a cheek cell and the resulting pH changes inside the said cell either directly or indirectly; the latter method preferably using a fluorescent probe specific for pH. The determination of 22 Na + uptake may be performed at a number of proton gradients ranging from about 10:1 to 100:1 (inside → outside). Differences in the net rate of proton-dependent sodium (Na + ) transport in the cheek cell and the response of the Na + uptake activity of the cheek cell to the imposed proton gradient, may form the basis for discriminating the hypertensive (adult) and/or pre-hypertensive (adolescent) individual, from others in the study groups, as will be described. Preferably, the epithelial cells are cheek epithelial cells, although other epithelial cells may be used as already indicated above. The advantages of using cheek cells are as follows: 1. Cheek cells are easily obtained from adults through to very young children and therefore the method suffers none of the problems inherent with blood sampling. 2. Cells can be obtained in a relatively non-invasive and entirely non-traumatic manner. Subjects may swish small amounts of water inside their mouth for a short period of time. The expectorate contains the "scuffed off cheek cells. 3. A biochemical assay can be done on each individual as sufficient cells can be obtained from one person over a period of about 3 minutes following the procedure outlined above and fully detailed hereinafter. As a result of the above points, the use of cheek cells in the method of the invention, would be expected to enjoy wide community acceptance in both general surveys and specific screening programs aimed at reducing the mortality and morbidity associated with hypertension. In yet a further aspect, the present invention provides an assay kit for use in the method of the invention, said kit including an indicator for detection of ion concentration in a suitable container, and an inhibitor of (Na + +K + )ATPase activity in a suitable container. The inhibitor may be ouabain. The indicator may be a fluorescent probe. The assay kit for use in the method of the invention may also include an appropriate mix of organic phthalates in a suitable container. The organic phthalate mixture may be used in a rapid centrifugation procedure whereby epithelial cells could be spun out of their 2 Na + -containing uptake solutions into the mixture. Detailed Description of the Assay Method In order to measure proton-dependent sodium transport it is necessary to subtract the background rate of non-proton dependent Na + uptake (passive diffusion) and to block the possible exit of Na + from the cheek cells by inhibiting (Na + + K + )ATPase activity (the sodium pump). It is necessary also to have an accurate measurement of cell number preferably by using cell protein content from each subject, in order to compare activities of proton-dependent Na + δ transport occurring in cheek cells of different individuals. The transport of Na + may be measured using radioactive sodium ions ^ 2 Na + ) after cheek cells are either acidified by incubating in pH 5.5 buffer (for proton- dependent 22 Na + uptake), or kept in pH 7.8 buffer (for non proton-dependent 22 Na + uptake) and then added to media of differing pH values to provide a range of proton gradient values. The proton-dependent and non-proton dependent rates of 22 Na + transport into cheek epithelial cells may be measured over various time points from as short as 15 seconds to as long as 40 minutes incubation at 25°C. For the study on adult hypertensive subjects to be described, uptake of 22 Na + was determined at 30 seconds and 5 minutes, while for the adolescent study to be described, uptake of 22 Na + was determined at 2 minutes and 5 minutes incubation. The uptake reaction is terminated and the cells containing ^Na* are collected by rapid filtration using 0.45 micron Mϋlipore filters. Trapped 22 Na + is counted by beta- liquid scintillation counting and the rates of 2 Na + uptake are compared on a milligram cell protein basis having determined cell protein content by the method of Lowry et al., (1951). A full description of the methods used for the recruitment of subjects for each of the studies described herein, the method of collection of the cheek epithelial cells, the method of measuring the Na + transport by the Na + /H + antiporter assay, the composition of all solutions pertinent to the said assay, the source of all chemicals and the methods used for the analysis of data, are given hereinafter. The present invention will now be described in reference to the accompanying figures. It is understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as it is outlined below. Brief Description of the Drawings Figure 1 illustrates the time course for sodium transport activity in cheek cells of a normotensive adult. The graph shows proton dependent and non-proton dependent rates at 22 Na + transport in cheek cells determined at indicated pH values (inside → outside) over a period of 40 minutes at 25°C, using the methods fully described in Appendix 1. Amiloride at a final concentration of ImM was tested at a proton gradient of 100:1 Figure 2 illustrates proton dependent (total), proton dependent + ouabain and non proton dependent (-fouabain) sodium transport rates in cheek cells of normotensive and hypertensive adults (30 sec incubation). Rates of 22 Na + uptake in cheek cells at a proton gradient (inside → outside) of 100:1 were determined as described in Appendix 2. Figure 3 illustrates sodium transport in cheek cells of normotensive and hypertensive adults (5 min incubation). Proton dependent (total), non proton dependent (control) and net proton dependent (total minus control) rates of 22 Na + uptake in cheek cells all, with ImM ouabain, were determined as described in Appendix 2. Figure 4 illustrates the effect of altered proton gradient on the net rate of proton dependent sodium transport in cheek cells of normotensive and hypertensive adults. Net rates of proton dependent (total minus control) 2 Na + transport at 5 minutes were determined at the indicated proton gradient (inside → outside) values as described in Appendix 2. Values for the regression coefficient (r) and the 95 % limits of confidence for each group are indicated. For the normotensive adult group, n=19 subjects were used for each proton gradient value. For the hypertensive adult group (n) values at each proton gradient were 17 (19:1); 15 (40:1); 15 (75:1) and 17 (100:1). Figure 5 illustrates sodium transport rates in cheek cells of normotensive and hypertensive adults. Net rates of proton dependent 22 Na + transport (total minus control) at 5 minutes incubation at a proton gradient value (inside → outside) of 75:1 were determined as described in Appendix .2. Figure 6 illustrates sodium transport rates in cheek cells of low BP and high BP tracking adolescents (2 min incubation). Proton dependent (total), non proton dependent (control) and net proton dependent (total minus control) rates of 22 Na + uptake in cheek cells at a proton gradient (inside → outside) of 100:1 were determined as described in Appendix 3. Figure 7 illustrates sodium transport rates in cheek cells of low BP and high BP tracking adolescents (5 min inculbation). Proton dependent (total), non proton dependent (control) and net proton dependent (total minus control) rates of 22 Na + uptake in cheek cells at a proton gradient (inside → outside) of 100:1 were determined as described in Appendix 3. Figure 8 illustrates time course for total and amiloride sensitive sodium transport in cheek cells of low BP and high BP tracking adolescents. Proton dependent total and proton dependent amiloride sensitive rates of 22 Na + uptake in cheek cells were determined at a proton gradient (inside → outside) of 100:1 over a period of 10 minutes as described in Appendix 3. Figure 9 illustrates the effect of altered proton gradient on the net rate of proton dependent sodium transport in cheek cells of low BP and high BP tracking adolescents. Net rates of proton dependent 22 Na + transport (total minus control) at 5 minutes were determined at the indicated proton gradient (inside → outside) values as described in Appendix 3. Values for the regression coefficient (r), and the 95% limites of confidnece for each group are indicated. Figure 10 illustrates sodium transport rates in cheek cells of individual low BP and high BP tracking adolescents, and high BP tracking adolescents with a family history of essential hypertension. Net rates of proton dependent 22 Na + transport (total minus control) at 2 minutes incubation at a proton gradient (inside → outside) of 32:1 were determined as described in Appendix 3. Figure 11 illustrates comparison of sodium transport rates in cheek cells of low BP and high BP tracking adolescents, and normotensive and hypertensive adults. Proton dependent (total), non proton dependent (control) and net proton dependent (total minus control) rates of 2 Na + uptake after 5 minutes incubation at a proton gradient (inside → outside) of 100:1 in cheek cells of low and high BP tracking adolescents and normotensive and hypertensive adults. Measurements of sodium transport were determined as described in Appendices 2 and 3. Characterisation of Human Cheek Cell Sodium Ion (Na + 1 Transport: (Na + /H + Antiporter Assay). The method of the invention in its preferred form relies on the fact that human cheek cells display significant sodium transport activity which by several criteria appears to be primarily due to the Na + /H + antiporter system. Although the Na + /H + antiporter system is known to be present in many mammalian cell types (see full reference list relating to Appendix 5), to our knowledge, there has been no reports in the scientific or patent literature that human cheek cells possess this activity. It therefore follows that the application of such a finding to the detection of a physiological abnormality such as the identification by biochemical means, of hypertensive and/or prehypertensive individuals is also unique. Human cheek cells display significant sodium transport (Na + uptake) activity, and, as a result of the various conditions tested in Figure 1, this activity is characteristic of Na + uptake via the Na + /H + antiporter system. The data shown in Figure 1, which is derived using the methods fully described in Appendix 1, shows that greatest Na + uptake occurs when cells are acidified at a pH of 5.5 and then Na + uptake is allowed to occur in pH 7.8 media. Under these conditions, this transient 100:1 outwardly-directed proton (H + ) gradient leads to significant Na + uptake (as measured by 22 Na + ). Uptake of Na + is greatly reduced when no proton gradient exists, e.g. pH 5.5^ -* 5.5 ∞t3iά . and pH 7.8 in , ide → 7.8 0Utside , or when an inwardly-directed proton gradient exists, e.g. pH 7.8;,,,^ → 5.5^^ 1 ^. This data strongly infers that Na + uptake is directly coupled to proton efflux and, as such, implies that the Na + /H + antiporter system can be activated in human cheek cells by the methods described. As will be shown hereafter, cheek cell Na + transport is also a function of the magnitude of the imposed (outwardly-directed) proton gradient, with the amount of Na + uptake generally increasing when the proton gradient is increased. (It is this response however which differs between the study groups investigated and forms part of the present invention). Nevertheless, this direct relationship between the magnitude of the proton gradient and the magnitude of the Na + uptake, further implicates the presence of Na + /H + antiporter activity in human cheek cells. Further evidence linking Na + transport activity in the cheek cell to Na + /H + antiporter activity, is provided by the results obtained with the specific Na + /H + antiporter inhibitor, amiloride. As is shown in Figure 1, addition of ImM amiloride to cheek cells activated to have a 100:1 outwardly-directed proton gradient, reduces Na + transport activity to levels comparable to that observed in cheek cells where no proton gradient exists. Two other characteristics of cheek cell Na + transport are evident from Figure 1. A steady-state level of Na + uptake is reached after about 10 minutes (probably corresponding in time to the fiill dissipation of the proton gradient), and an "overshoot" or decrease in the rate of the Na + uptake occurs after that time. Both of these observations are characteristic of the profile of Na + /H + antiporter activity obtained in other cell types (see for examples references in Appendix 5) and further implicates the Na + /H + antiporter as being the predominant system involved in Na + transport in the human cheek cell. The assay conditions for determining cheek cell Na + transport activity which were routinely used in the studies to validate this invention, are modified from existing methods for assaying Na + /H + antiporter activity present in other tissues and cell types (as indicated in Appendix 1). Our modifications are based on empirical observations using cheek cells which were made during the early course of the studies described herein , and no attempt is made to exhaustively inves¬ tigate all the characteristics of human cheek cell Na + transport and the role of the Na + /H + antiporter in this transport system. Not withstanding this, the following observations have been made with respect to Na + transport and Na + /H + antiporter activities in human cheek cells, and these have hitherto not been previously reported: Rates of Na + uptake by human cheek cells are comparable to the reported rates of Na + uptake (via the Na + /H + antiporter system) in other cell and tissue types as outlined in Appendix 5, even when allowance is made for the wide variety of assay methods employed in the various studies listed. The final concentration of Na + in all the cheek cell assays de- scribed in this invention, is 1 mM. As such, this concentration is well below the K,„ (Michaelis-Menton constant) for the cheek cell Na + /H + antiporter system which is between 6mM and lOmM Na + for a normotensive adult subject. - The K,,, for the internal proton binding site of the Na + /H + antiporter of cheek cells from a normotensive adult subject is about 1.7μM (H + ). The Hill coefficient of this site approximates 1. Cheek cell Na + /H + antiporter activity is not greatly affected by the K + ionophore, valinomycin, indicating that cell membrane potential does not greatly influence Na + transport activity. As such, this is in agreement with the findings reported in the scientific literature for Na + /H + antiporter activity in other cell and tissue types. - The extent of Na + transport in cheek cells having an outwardly- directed proton gradient of 100:1, is many fold higher in the presence of the ionophores monensin (Na + : H + ) or nigericin (K + : H + ). Such findings also validate the existence of Na + /H + antiporter activity in human cheek cells. The transport of Na + into cheek cells following the activation of 5 the Na + /H + antiporter system by an outwardly-directed proton gradient, represents the net transport of the Na + ions, which is, in turn, the sum of the rate of Na + uptake and the rate of Na + efflux. Efflux of Na + from the cheek cell occurs simultaneously to, and is dependent on, the activation of the Na + /H + antiporter system and 10 the resulting increase in cheek cell 22 Na + content. However, up to a time of 10 minutes, Na + influx exceeds Na + efflux. The rate of Na + efflux is also time-dependent and hence dependent on the extent of uptake of Na + into the cheek cell which has occurred via the Na + /H + antiporter. The "overshoot" phenomenon mentioned 15 previously, may represent a situation of greater Na + efflux than influx after complete dissipation of the proton gradient and subse¬ quent deactivation of the Na + /H + antiporter activity. The contribution of other known Na + transport systems to the net 20 transport of Na + in human cheek cells is not great. Efflux of (transported) 22 Na + out of the cheek cell via the Na + +K + -ATPase (sodium pump), does affect the net rate of Na + influx to a small but significant extent (see Figure 2). However, to entirely eliminate any contribution by this Na + transport pathway to net cheek cell 25 Na + transport activity, ImM ouabain, a specific inhibitor of the sodium pump, is routinely included in all those assays concerned with the studies that validate this invention (as indicated in the appropriate Appendices). On the basis of inhibitor studies using bumetanide and frusemide, the contribution of the Na + /K + /2C1 " 30 cotransport system to the overall Na + transport activity of human cheek cells, is insignificant. Human cheek cells also contain a significant level of Na + /Li + antiport activity in which Na + uptake into cells is coupled to an outwardly directed Li + gradient. In conclusion, the Na + transport activity of human cheek cells, which is used to validate this invention, has only partly been characterised at the biochemical level. However it is now firmly established that the Na + /H + antiporter system is largely responsible for the observed Na + transport. All the abovementioned characteristics of Na + transport in human cheek cells therefore represent new and unique findings not previously published in the scientific or patent literature. The discovery of the potential for human cheek cells to transport Na + in a predictable manner, forms the basis for the conclusion that hypertensive and prehypertensive individuals can be identified using a biochemical marker. These specific details of the invention will now be described. Adult Cheek Cell Study Subjects used in this study were recruited from adult volunteers identified following several blood pressure screening programs. None of the control or hypertensive subjects were receiving anti-hypertensive medication at the time of the blood pressure screening or sampling of cheek cells. All measurements were done using a Dinamap (automatic blood pressure recording instrument). Those first identified as being mildly hypertensive (diastolic blood pressure > 95mm Hg), were subsequently re-measured over a period of several days to confirm the reading. Systolic and diastolic blood pressures, age and gender of the two groups used in this example are shown in Table 1. Systolic and diastolic blood pressures were significantly different between the two groups, while the groups were well matched for age and gender, although the hypertensive group exhibited a slightly greater body mass index (BMI). Table 1 CHARACTERISTICS OF NORMOTENSIVE AND HYPERTENSIVE SUBJECTS USED IN THE CHEEK CELL ASSAY FOR HUMAN HYPERTENSION BLOOD PRESSURE (n) SYSTOUC DIASTOLIC AGE M/F %F BMI (mm Hg) (mm Hg) (years) (Kg/m 2 ) 1 Data is presented as the mean ± SEM for the indicated number of subjects (n) in each group. Differences between means were determined by Student's unpaired t-test significant. 2 For proton gradients perfoπned at 19: 1 , 40: 1 and 75: 1. Cheek cells were isolated from all subjects in the morning and assayed in the afternoon of the same day as collection. Proton-dependent sodium transport was determined using triplicate assays of both the total sodium uptake rate and the non proton dependent rate to determine the proton-dependent sodium uptake rate, as described in the appropriate methods of Appendix 2. The variability of the measurements across all assay conditions for any one individual averaged 6.2% (interassay variation), while for assays on the same subject(s) performed on different days, the variability averaged 10.6% (intra-individual variation). Figure 2 compares Na + transport in cheek cells of normotensive and hypertensive adult study groups after the Na + transport assay has been allowed to proceed for 30 seconds. As indicated in the legend to this figure, the total rate of Na + uptake is that observed when an outwardly-directed proton gradient of 100:1 is applied to cheek cells. Inclusion of ImM ouabain, a specific inhibitor of (Na + +K + )-ATPase activity, results in a small increase in Na + uptake in both normotensive and hypertensive adult subjects. As discussed in the previous section, this result suggests that the contribution of the sodium pump to Na + efflux, is minimal. Ouabain (at ImM) is added to all assays in which Na + transport activity of adults and adolescent study groups is investigated to completely negate any contribution from the sodium pump. It is also clearly apparent from Figure 2, that the total and total plus ouabain rates of Na + uptake in cheek cells of the adult hypertensive study group is only about 50% of that evident in cheek cells from the normoten¬ sive study group. Furthermore, this difference in Na + uptake rates for the above conditions is highly significant between the two study groups. For hypertensive adult subjects, the control rate of Na + uptake in the presence of ouabain, (which represents the rate of "passive" Na + uptake into cheek cells in which Na + /H + antiporter activity has not been activated by a proton gradient), is also much lower than that for the normotensive study group, but statistical significance is not achieved. Figure 3 compares Na + transport in cheek cells of normotensive and hypertensive adult study groups after the Na + transport assay has been allowed to proceed for 5 minutes. It is clear that the total and the net rates of Na + uptake for the hypertensive study group are only about 50% of that evident for the normotensive study group. Furthermore, this difference in the (proton-dependent) Na + uptake rates is highly significant between the two study groups. The control (non-proton dependent rate) of Na + uptake is not significantly different between the study groups. The difference in cheek cell Na + transport activity between normotensive and hypertensive study groups is clearly apparent only under conditions which would lead to activation of the Na + /H + antiporter system, i.e. the presence of an outwardly-directed proton gradient. This fact indicates that intrinsic differences in the activity of the Na + /H + antiporter system and/or its activation by a proton gradient, may underlie the dramatic difference seen in the Na + transport activity between the normotensive and the hypertensive study groups. This has been further investigated in cheek cells obtained from the respective adult study groups, by comparing the response of the Na + /H + antiporter system (and hence Na + uptake), to outwardly-directed proton gradients which differ in their magni¬ tude. The response of cheek cell proton-dependent sodium transport to proton gradients varying in magnitude from 19:1 to 100:1 (inside → outside) in the normotensive and hypertensive study groups is shown in Figure 4. A clear distinction is apparent between the two groups. Not only is the rate of proton-dependent sodium transport in the hypertensive subjects markedly reduced at all proton gradient values as compared to the normotensive group, but for the hypertensives, the rate of proton-dependent sodium transport is unaffected by a change in proton gradient values from 19:1 to 75:1. Only at a proton gradient value of 100:1 does the rate of proton-dependent sodium transport of the hypertensive group increase above the value exhibited for proton gradient values of 19:1, 40:1 and 75:1. The difference in the rate between the two groups is extremely significant for all but the 19:1 value of the proton gradient. The rate of proton-dependent sodium transport in cheek cells as a result of imposing a proton gradient of 75:1 (inside → outside) for 15 individual hyperten¬ sives and 19 normotensive subjects, is shown in Figure 5. [A proton gradient of 75:1 refers to the ratio between the hydrogen ion concentration in the cell loading buffer and the assay (uptake buffer) respectively, after taking into account the dilution effects arising as a result of the manner in which the transport assay is performed. For a 75:1 proton gradient to be achieved, the cell loading buffer has a value of pH 5.5 and the assay uptake buffer a value of pH 7.65]. Cheek cells from the 15 hypertensive subjects display a markedly reduced mean rate of proton-dependent sodium transport (mean ± SEM; 0.41 ± 0.1 nmol Na +# mg protein»5min) which is 34% of the rate for the 19 normotensive subjects (1.20 ± 0.16 nmol Na + »mg protein # 5min), and this difference is highly significant (P=0.0004; Student's unpaired t-test). Some overlap between the values for hypertensive and normotensive subjects is apparent in this data (approximately 6% as defined in Table Appendix 6). However as discussed in relation to Appendix 6, this degree of overlap is less than that obtained in any previous study where the rates of cation transport in other cell types have been measured in relation to hypertension. The response of cheek cell proton-dependent sodium transport to a change in the value of the proton gradient from 19:1 to 75:1, is shown in Table 2. These values represent the slope of the line between the abovementioned proton gradient values as depicted in Figure 4. As can be seen from this table, the slope of the line between the normotensive and hypertensive subjects differs by a factor of 24 fold at a significance level of P = 0.0028; i.e. the mean increase in proton- dependent 22 Na + transport is 24 times greater in the normotensive subjects as compared to the hypertensive subjects. For the above parameter of proton- dependent sodium transport, the extent of overlap between the values for the individual normotensive and hypertensive subjects is 11.5% as defined in Appendix 6. Table 2 RESPONSE OF CHEEK CELL PROTON-DEPENDENT SODIUM TRANSPORT TO A CHANGE IN THE PROTON GRADIENT IN NORMOTENSIVE AND HYPERTENSIVE ADULT SUBJECTS Normotensive n=19 0.48 ± 0.1 1 nmol 22 Na # mg protein » 5 min (for a change in the proton gradient from 19:1 to 75: 1) Hypertensive n=15 0.02 ± 0.1 Significance P=0.0028 1 Data is presented as the mean ± SEM for the indicated number of subjects (n) in each group. The significance of the difference between the mean was determined by Student's unpaired t-test. The present invention has significant potential in the determination of human hypertension. Marked differences in the rate of proton-dependent 22 Na + transport into cheek cells are apparent between age and sex-matched hypertensives and normotensive subjects under a wide range of assay conditions. However when the assay is performed at a proton gradient value of 75:1 and measured over a period of 5 minutes, the differences between normotensive and hypertensive subjects is most pronounced, (mean value of proton-dependent 22 Na + uptake in the hypertensive group is only 33% of that evident in normotensive group); of highest statistical significance, (P value of 0.0004 between the two groups, i.e. 1 chance in 2,500 that the result occurred by chance alone); and capable of the greatest discrimination between the two groups (i.e. where least overlap occurs; average 6% as defined in Appendix 6. The increase in the rate and amount of proton- dependent 22 Na + uptake when the proton gradient is increased from a value of 19:1 to 75:1 is also a useful pan leter to discriminate between hypertensive and normotensive subjects. Adolescent cheek cell study The preceding sections have clearly demonstrated that: 1. Human cheek cells have the ability to transport Na + primarily via proton gradient activation of an associated Na + /H + antiporter system; 2. There is a statistically significant reduction in the magnitude of Na + transport activity in cheek cells from adult hypertensive subjects com- pared to adult normotensive subjects; 3. The difference in Na + transport activity between these adult study groups appears related to the proton-gradient dependent cheek cell Na + /H + antiporter system. In order to extend these findings and test one of the claims of this invention, i.e. that the biochemical parameter under study viz-a-viz Na + transport in cheek cells, can be used to identify pre-hypertensive individuals, a study using adolescents is described. Full details of the methods used in the Adolescent cheek cell study are described in Appendix 3. Based on the previously reported finding that the tendency during childhood for blood pressure to track in an upper or lower percentile is reason¬ ably strong, and that it is a good predictor of the adult ranking, adolescents who consistently tracked in an upper and lower percentile rank, were identified from blood pressure readings taken over a period of at least four years. Details of those adolescents who were finally selected for this study are described in Table 3. The low blood pressure (BP) tracking group of n=24, represents 4.8% of the total group of n=504 adolescents who were screened. Subjects within this group consistently exhibited the lowest systolic BP reading from the commencement of the study in 1986 to the time at which cheek cell Na + transport activity was determined. In contrast, the high BP tracking group, representing 5.7% of the total group screened, consistently exhibited the highest (systolic) BP readings of all the subjects screened. (Statistical procedures used to determine the BP ranking status of these two groups of adolescents are fully described in Appendix 3). It can be clearly seen from the data in Table 3, that the difference in the systolic BP between the two adolescent groups remained significantly different (P< 0.0001) over the four-year period of the tracking study. No significant difference in the age or BMI of the two groups is evident at the time the cheek cells were actually sampled. However, of particular importance is the extent by which the two adolescent groups differ in their incidence of a definitive family history of hypertension. In the high BP tracking group, 7 of the 29 subjects (24%) had a definitive family history of hypertension with one or both of the parents being treated with a recognised anti-hypertensive medication. This contrasts with the low BP tracking group where a transient secondary hypertension (eclampsia) was evident for the mother of one of the subjects. A higher proportion of males (69%) is present in the high BP tracking group in comparison to the low BP tracking group, in which 50% were male. Table 3 SYSTOLIC BP (1986-87) 100 ± 0.7 123 ± 0.8 < 0.0001 23mm Hg SYSTOLIC BP (1989-90) 105 ± 0.9 129 ± 1.0 < 0.0001 24mm Hg (NS) Not significant It must be emphasised that these groups are not directly comparable to the normotensive and hypertensive adult study groups referred to in the previous section. For example, none of the high BP tracking adolescents recorded BP readings which impinged on values characteristic of mildly adult hypertensive subjects. However, each group displayed consistently different BP tracking behaviour over a period of 4 years up to the time of cheek cell sampling. As such, it would be expected that a far greater proportion of those subjects within the high BP tracking group may later develop hypertension compared to the low BP tracking group. This prediction is further reinforced by the observation that 7 of the high BP tracking adolescents had a definitive family history of hyperten¬ sion. The higher proportion of males and the slightly higher BMI of subjects within the high BP tracking group, (both of which are characteristics of adult essential hypertension), is probably only a co-incidental finding in this study. Cheek cells from adolescent subjects from each BP tracking group were assayed for Na + transport activity ^ 2 Na + uptake) on the day of isolation using the methods fully described in Appendix 3. Figure 6 shows the total, control and net rates of Na + transport activity following a 2 minute assay time course for cheek cells from subjects within the two adolescent study groups. Both proton-depend- ent (total and net) and non-proton dependent (control) rates of Na + transport, are significantly reduced in cheek cells from subjects in the high BP tracking group. The extent of this reduction in cheek cell Na + transport activity (about 50% of the rate apparent for the low BP tracking group), equates with the extent of the difference evident between adult normotensive and hypertensive study groups (described in the previous section). Figure 7 shows the total, control and net rates of Na + transport activity following a 5 minute assay time course for cheek cells from subjects within the two adoles¬ cent study groups. As is the case for the 2 minute time point, both proton- dependent (total and net) and non-proton dependent (control) rates of Na + transport, are significantly reduced in cheek cells from subjects in the high BP tracking group. Again this equates with the extent of difference evident in the adult cheek cell study. The only distinction between the adult and adolescent studies thus far, is the difference in the rate of Na + transport activity in the control assay situation where the absence of a proton gradient would be expected not to activate Na + /H + antiporter activity. In the adolescent study this difference does reach statistical significance. However a similar trend of lower (control) Na + transport activity in cheek cells from subjects in the adult hypertensive study group, is clearly apparent. The effect of amiloride, a specific inhibitor of Na + /H + antiporter activity, has also been tested in the adolescent study. The rationale for including this addition¬ al step in the assay protocol is to determine whether the amiloride sensitive Na + transport rate, which represents that component of Na + uptake dependent on the activity of the Na + /H + antiporter, differs between the two adolescent study groups. Figure 8 shows a time course for the total and the amiloride-sensitive rates of Na + transport in cheek cells from subjects in the low and high BP tracking groups. Both total and amiloride sensitive Na + transport activity in cheek cells from subjects in the high BP tracking group are significantly reduced in comparison to the low BP tracking group at all four time points examined. This result further indicates that some property of the cheek cell Na + /H + antiporter activity and/or its activation by a proton gradient, underlies the phenomenon observed in these studies. As was done in the adult study, the activation of the Na + /H + antiporter system by a proton gradient has also been investigated in cheek cells obtained from the respective adolescent study groups. This was accomplished by comparing the response of the Na + /H + antiporter system (and hence Na + uptake), to outwardly-directed proton gradients which differ in their magnitude. The response of cheek cell proton-dependent sodium transport to proton gradients varying in magnitude from 10:1 to 100:1 (inside → outside) in low BP and high BP tracking adolescents is shown in Figure 9. The rates of proton-dependent sodium transport in the high BP tracking adolescent group are markedly reduced at all proton gradient values as compared to the other adolescent study group. Furthermore, for the high BP tracking groups, the slope for the proton gradient activation of cheek cell Na + transport is markedly reduced over that obtained for the low BP tracking group. The response of the cheek cell proton dependent sodium transport to a change in the magnitude of the proton gradient from 10:1 to 100:1, is shown in Table 4. These values represent the slope of the line between the abovementioned proton gradient values as depicted in Figure 9. As can be seen from this table, the slope of the line between low and high BP tracking adolescent groups differs by a factor of 2.5 fold at a significance level of P=0.002; i.e. the mean increase in proton-dependent Na + transport is 2.5 times less in the high BP tracking adoles¬ cents as compared to the low BP tracking adolescents. However, the magnitude of this difference in the slope of the proton-dependent Na + transport between the adolescent study groups is nowhere near the 24 fold difference evident in the adult study. The fact that the magnitude of this difference in the adolescent study was "dampened" as compared to the adult study, may be a reflection of the different nature and composition of the two study groups in question. Figure 10 shows sodium transport rates (at a proton gradient value of 32:1) in cheek cells of individual low BP and high BP tracking adolescents, and high BP tracking adolescents with a family history of essential hypertension. Cheek cells from the high BP tracking group display a markedly reduced mean rate, (mean ± SEM; 0.73 ±0.08 nmol Na + »mg protein«5min) of proton-dependent Na + transport which is 57% of the rate for the low BP tracking adolescents; (1.28 ±0.17 nmol Na +β mg protein β 5min), and this difference is highly significant (P = 0.0032; Student's unpaired t-test). The mean Na + transport rate for those 7 subjects in the high BP tracking groups (0.46±0.07 nmol Na +β mg protein»5min) is 63% when compared to the mean rate for the total high BP tracking adoles¬ cents, and 36%; (P=0.016) when compared to the mean rate for the low BP tracking adolescents. Table 4 RESPONSE OF CHEEK CELL PROTON-DEPENDENT SODIUM TRANSPORT TO A CHANGE IN THE PROTON GRADIENT IN LOW BP TRACKING AND HIGH BP TRACKING ADOLESCENT SUBJECTS Low tracking n=24 0.91 ± 0.15 1 nmol 22 Na # mg protein»5 min (for a change in the protein gradient from 10:1 to 100: 1) High tracking n=29 0.36 ± 0.09 Significance P=0.002 1 Data is presented as the mean ± SEM for the indicated number of subjects (n) in each group. The significance of the difference between the mean was determined by Student's unpaired t-test. The Na + transport rates of high BP tracking adolescents with a family history of hypertension are all below the mean value for Na + transport for the total high BP tracking group, and overlap with only 3 of the 24 subjects in the low BP tracking group. It is therefore clearly apparent that those adolescents who display both a high BP tracking profile together with a family history of hypertension, also have the characteristic of a significantly and distinctly low rate of proton-dependent Na + transport activity in their cheek cells, and indeed can be distinguished by this latter characteristic. In this regard, the biochemical parameter under study in this embodiment, i.e. cheek cell Na + transport activity, displays similar characteristics in this group of adolescents (who may be considered at greatest risk of later developing hypertension) when compared to the group of hypertensive adults described in the previous section. Finally, a comparison is made between the sodium transport rates of cheek cells from the study groups within the adolescent and the adult studies. Figure 11 shows the total, control and net rates of Na + transport in the adolescent and adult studies when examined after a 5 minute assay time course and a 100:1 outwardly- directed proton gradient. The extent of the difference between low BP and high BP tracking adolescents on the one hand, and between normotensive and hyper¬ tensive adults on the other, is striking for all three assay variations examined. Despite the fact that there was a six-month interval between these studies, with the adolescent study following after the adult study, the overall rates of cheek cell Na + transport activity are nevertheless very similar. Discussion A comparison of previous studies of cation transport in red blood cells, leucocytes and platelets of hypertensive and normotensive subjects with the present cheek cell assay for hypertension, is shown in Appendix 6. Twenty three other studies, most of which have used red blood cell Li + -Na + countertransport activity to discriminate between hypertensive and normotensive subjects, are listed in this Appendix. For ease of comparison of these data with that of our own, some computational parameters have been calculated to allow both the extent of the difference between the normotensive and the hypertensive rates of cation transport (Hx/Nx) and the extent of overlap between the two groups, to be more readily compared; (these terms are defined in the legend to Appendix 6). It is immediately apparent from our data on the adult hypertensive study that unlike the situation in all the other cited studies, the rate of proton-dependent sodium uptake is lower in hypertensive subjects when cheek cells are used for measurement. This contrasts with the six studies in which Na + /H + antiporter activity has been measured in red blood cells, leucocytes and platelets. Li + -Na + countertransport activity in red blood cells, which is most often measured as the rate of sodium-dependent lithium efflux, is more difficult to relate to the Na + /H + antiporter of proton-dependent sodium transport. Nevertheless, in the studies shown, Li + -Na + countertransport activity in red blood cells of hypertensive subjects is between 104% and 371 % the rate observed with normotensive subjects and the significance of the difference between the two groups ranges from N.S. to P<0.001. This same situation of decreased sodium transport activity is also apparent in the adolescent cheek cell study. For the adolescent study however, the extent of overlap has not been included or compared to the other studies shown in Appen¬ dix 6 for the previously stated reasons that the adolescent study groups are not directly comparable to the studies using adult hypertensive and normotensive subjects. Figure 11 showing a comparison of cheek cell sodium transport activity between the adult and adolescent studies confirms the similarity of the two studies reported in this invention. When these combined results are viewed against the studies of others shown in Appendix 6, it is clear that the parameter measured in the cheek cell assay is behaving in a manner opposite to that reported by others (using other cell types) in relation to hypertension (and also the prehypertension situation). The converse finding of a lower cation transport activity in the cheek cells of our hypertensive group, (and in the BP high tracking adolescent group with a family history of hypertension; the "prehypertensive group") is at present difficult to explain in the light of the findings from the cited studies. However, in this respect we have noted that cheek cells exhibit certain properties which differ significantly from those found in the other types of cells cited in Appendix 6. One example is the apparent inability of cheek cells to display significant osmotic behaviour as is the case for human red blood cells. This would suggest that a basic difference may exist in cell membrane permeability to ions and solutes in cheek cells as opposed to many other mammalian cell types. It is therefore possible that some property unique to epithelial cells may account for our particular observation in relation to the hypertensive and normotensive groups as well as in the adolescent study. One likely explanation of our result is that the cheek cell plasma membrane of the adult hypertensive and the "prehypert¬ ensive" group, has a far greater permeability to protons (H + ) than is the case for the other two comparable study groups. This would imply that with the imposition of a proton gradient (inside → outside), less protons would be available to couple with the cell membrane located Na + /H + antiporter due to increased loss of cytosolic protons through the cell membrane; hence the finding of a lower rate of proton-dependent sodium transport in these (hypertensive/prehypertensive) groups. An increase in cheek cell membrane permeability to protons in the hypertensive and prehypertensive groups is borne out experimentally by examining the response of cheek cell sodium transport activity to proton gradients differing in their magnitude. It is clear from the data in Figures 4 and 9, and Tables 2 and 4, that the slope (or response) of sodium transport to an increasing proton gradient is significantly less in both the above study groups across the range of proton gradient values examined. This would indicate that an equivalent number of protons is either not present (via rapid dissipation due to increased membrane permeability), and or that the extent of coupling of proton efflux to sodium uptake differs in the hypertensive (and prehypertensive) groups compared to their respec¬ tive control groups. Besides a difference in membrane proton permeability and/or proton coupling to the cheek cell Na + H + antiporter, yet another explanation for over result may lie in an enhanced permeability of the cell membrane to sodium ions, particularly in regard to the rate of efflux of Na + which has entered the cheek cell during the proton activation of the Na + /H + antiporter. We have observed that efflux of 22 Na + from cheek cells is a rapid event and that Na + efflux is probably occurring during the Na + uptake process, albeit at a rate less than the rate of Na + uptake during the first 10 minutes of the assay incubation when measured at 25°C. A greater rate of efflux of transported Na + ions in the cheek cells of the hyperten¬ sive and prehypertensive groups compared to their respective control groups, could account for our experimental findings. An important difference between the cheek cell assay method described herein and those studies of others referred to in Appendix 6, is the final concentrations of sodium ion (Na + ) used in the respective assays. In contrast to the studies listed in Appendix 6, all of which used a final concentration of about 150mM Na + and did not rely on a radioisotopic technique for assay, the present invention uses a final concentration of ImM Na + containing 4.3 to 6.4 μCi (159-237 kBq) 22 Na + per μmole Na + . As has been previously mentioned in the section dealing with the characterisation of cheek cell Na + transport and Na + /H + antiporter activity, a final concentration of ImM Na + is well below the Km value of Na + for the cheek cell Na + /H + antiporter. As such, in our assay this transport system would be operating with Na + transport rates many times less than the maximum rate achievable if Michaelis-Menton kinetics were operative and the substrate (Na + ) was saturating. This is in direct contrast to the situation in the other studies listed in Appendix 6 in which the concentration of Na + in those assays would be saturating and the Na + /H + antiporter would be operating at its maximum rate. As a consequence of this important difference in the manner in which the sodium transport assays were carried out between this and the other cited studies, it would be expected that the respective assays would exhibit a differential sensitivi¬ ty to those parameters which may affect the rate of sodium ion transport by the Na + /H + antiporter. Thus, under saturating (high Na + assay concentration) condi- tions, differences in the absolute amount of Na + /H + antiporter catalytic unit(s) present, may be more readily observable than would be the situation when substrate is limiting as is the case in the presently described cheek cell assay. On the other hand however, at low substrate (Na + ) concentrations where the activity of the Na + /H + antiporter is well below its maximum rate, the influence of other factors, such as proton permeability may be more apparent. This may come about due to the fact that the outwardly-directed proton gradient is not being rapidly dissipated via the Na + /H + antiporter because of the latter' s relatively low activity. Dissipation of protons (H + ) could then occur via other mechanisms which may involve H + permeation through the cell membrane, and this rate of H + dissipation may represent an intrinsic and heritable characteristic of human essential hyper¬ tension. As such, the cheek cell assay method described herein may be inherently more sensitive to those biochemical factors which are perturbed in hypertension in comparison to the methods of others described in Appendix 6 which neither use cheek cells nor the assay techniques described in this invention. In comparison to the studies cited in Appendix 6, it is clearly evident that for the adult cheek cell study, the difference in cation transport activity between normo¬ tensive and hypertensive subjects (irrespective of which group has the greater rate), exhibits the following: (i) the greatest magnitude; Nx/Hx = 2400% (ϋ) the greatest statistical significance; P< 0.0004 (iϋ) the least overlap; 6%. With reference to (ϋi): Overlap has been arbitrarily defined in Appendix 6 and data to calculate this parameter is only available in a limited number of studies. Attention is drawn to the Canessa study (Canessa et al., 1980) in which an overlap value of 0% was first reported. In their follow-up study (Semplicini et al., 1989) in which identical methods to the Canessa et al., (1980) study were employed, the overlap value averaged 26%. The studies outlined in Appendix 6, particularly those in which Na + /H + (rather than Li + -Na + ) countertransport activities have been determined, do not show as great a difference between the two groups (141 % to 195 %) as found using the cheek cell assay method (293% to 2400%;Nx/Hx). This finding may be due to the fact that this present study was unique in that a range of proton gradient values were investigated with respect to proton-dependent sodium transport and that this present assay concentration of Na + was 1 mM. Conclusions It will be seen that the present invention is significant in that it achieves the following: 1) It allows the activity of a biochemical marker to be correlated with physiological disorders such as human hypertension in cheek cells which can be obtained in a relatively non-invasive manner. 2) It allows the potential for screening those people in the community who may be genetically predisposed to develop hypertension but are still in the prehypertensive state. Included in this category would be those adolescents who have high BP tracking characteristics and a family history of hypertension. 3) It is noted that this latter property is particularly significant in that for those who are identified as being in the prehypertensive stage, early measures could be taken to ameliorate later development of hypertension, or, at the very least, to minimise the extent of the hypertensive disorder. This invention provides an assay which can be used to detect differences in the activity of a biochemical marker present in epithelial cells which can, with a minimum of overlap and with high statistical probability, discriminate between adult hypertensive and normotensive subjects, and between adolescents who have a family history of hypertension and high blood pressure tracking characteristics and those adolescents who do not. For the adult, the invention achieves a level of discrimination which is greater than any existing test done with cells derived from the blood of hypertensive subjects. For the adolescent, no such biochemical test has been described in the scientific literature to which the present invention can be compared. Finally, it is clearly apparent from the examples given, that we have developed a technique which is far less invasive, and has greater discriminatory ability than all other tests so far described in the prior art. Modifications to the Assay Method The transport of sodium ions (Na + ) could also be measured using a fluorescent probe sensitive to small changes in Na + concentration in the cytoplasm of cheek cells resulting from Na + /H + antiporter activity. Such a probe (SBFI/AM) is readily available commercially. The methodology for using this probe to measure changes in cytosolic Na + concentration in cells other than cheek cells, and in a variety of situations, has been described in the scientific literature. Proton-dependent sodium transport and changes occurring in this parameter in association with hypertension, could also readily be measured by following the movement of protons (H + ) into and out of cheek cells. Movement of H + will result in changes in cheek cell cytosolic pH and this can also readily be measured by using a fluorescent probe sensitive to small changes in cytosolic pH in the cheek cell. Such a probe (BCECF/AM), together with a variety of other pH- sensitive probes, are also available commercially. Again, as for the Na + sensitive probe, there is comprehensive literature related to the general use of such fluores¬ cent probes. The fluorescent probe method(s) either singularly or in combination would greatly increase the speed at which assays could be done. They have the advantage over the radioisotopic method of providing a continuous reading of Na + and/or H + concentrations which would therefore turn the assay into a kinetic one. Measure- ments of proton-dependent sodium uptake (or sodium-dependent proton efflux) could be done using a suitable spectrofluorimeter which is available commercial¬ ly. The assay may be designed in a manner similar to the radioisotopic method. Human cheek cells would be acidified by the methods described in this invention. The change in Na + concentration or cell pH (H + concentration), following the imposition of a suitable proton gradient, would be directly measured by following the change in the fluorescent spectra of the respective fluorescent probes(s). The above methodology could also be applied to directly measure the intrinsic Na + and H + permeability of the cheek cell to these ions. That is to say, that if the phenomenon described in this invention is explainable by an underlying biochem- ical mechanism involving an intrinsic difference in the permeability of the cheek cell membrane to these ions, then this permeability difference could also be measured. For example, in the final assay it may only be necessary to determine the response of the cytosolic pH of the cheek cell to changes in the external pH; i.e. to determine cytosolic buffering capacity in hypertensive and pre-hypertensive subjects. A fluorescent probe sensitive to cell pH such as BCECF/AM, could also be used in this regard. It is also quite conceivable that the fluorescent probe method could subsequently be simplified to a far greater extent than described above. For example, the change in cheek cell Na + or pH (H + ) concentration could be detected at a single time point after the mixing of the reagents was completed. This may only involve a single point reading using a commonly available low-cost spectrofluori¬ meter. Further streamlining the assay procedure described in this invention to reduce cost, increase throughput and increase sensitivity, could easily be done by modifying the methods used in the assay of the biochemical marker as previously described. One such modification could be to rely on gamma counting as opposed to liquid scintillation counting as is done in the presently described invention. Sodium-22 has the following characteristics with regard to the emission of ionising radiation: Eβ, 0.546 Mev; Eγ, 1.275 Mev. Gamma count- ing would make use of the relatively strong γ-ray emission and would negate the use of scintillation fluid in the counting step. Another such modification would be to separate cheek cells from their uptake media using rapid centrifugation procedures in contrast to the rapid Millipore filtration method described in the present invention. Cheek cells could be spun out of their 22 Na + -containing uptake solutions using 1.5ml Eppendorf centrifuge tubes loaded with an aliquot of a suitable density mix of phthalates into which cheek cells would migrate but the aqueous 22 Na + -containing uptake solutions would not. As mentioned previously, the kit of the invention may include an appropriate mix of organic phthalates in a suitable container. The kit may also include a protocol for a centrifugation step to replace the Millipore filtration step in said assay procedure. Gamma counting can be used to replace beta liquid-scintillation counting. Other Uses to which Human Cheek Epithelial (Buccal Mucosal) Cells have been Applied. Although cheek cell scraping from the mouth is used extensively in teaching aspects of biology and cell structure in course work, to our knowledge their use in more applied scientific research did not occur until 1984. At that time the principal investigator concerned with the invention described herein, made use of human cheek cells to examine lipid profiles in dietary and nutritional studies. These studies were reported in the following publications which have been fully listed in the bibliography. McMurchie et al., 1983; McMurchie et al., 1984a; McMurchie et al., 1984b; McMurchie et al., 1984c; Rohan et al., 1984; Margetts et al., 1985. Since establishing the potential for human cheek cells to offer a relatively non- invasive source of tissue, a variety of different studies on cheek cells have appeared in the literature (it is stressed however, that to our knowledge, no studies concerning hypertension in general, or ion transport in particular, have appeared). These studies have been fully listed in the bibliography and include the following: Badcock et al., 1986; Tamai et al., 1988; Lench et al., 1988; Sampugna et al., 1988 and Wang et al., 1990. In addition, the following articles have made reference to the use of cheek cells in nutritional studies: Relation of changes in dietary fatty acid to alterations in linoleic acid content of human cheek cell phospholipids. Editorial Review, Nutrition Reviews (1984), 42 376-377; (EJM work cited in references): Methods for obtaining fat microbiopsies. Editorial comment, Nutrition Reviews (1986), 44, 200-201; (EJM work cited in references). Appendix 1 Materials and methods for measuring the characteristics of sodium transport in human cheek cells. Collection of cheek cells Cheek (buccal mucosal) cells were obtained in a non-invasive manner by having subjects swish distilled water around their mouth for a short period of time, using a gentle molar scraping action. The expectorate containing the cheek cells was collected and a further two or three 10ml washes produced an average total yield of about 4 million cells. Samples were spun at 46,000g x 15 minutes in a Beckman J2-21 centrifuge using a JA-20 rotor at 4°C. The packed cheek cell pellet was resuspended in 1ml distilled water by gentle homogenisation. Cheek cells were isolated from subjects in the morning and assayed for Na + /H + antiporter activity on the same day. Measurement of Na + transport by the Na + /H + antiporter assay Na + /H + antiporter activity was measured by the uptake of 22 Na + using a modification of the Millipore filtration technique as described by Seiler et al., (1985). All cell equilibration and transport buffers used were prepared by titrating a pH 5.5 buffer: 230mM mannitol, 49mM MES, 11.2mM N-methyl-D-glucamine (NMG), ImM ethyleneglycol-bis-(|8-amino-ethyl ether)N,N'-tetra-acetic acid (EGTA), with a pH 8.4 buffer: 230mM mannitol, 16.8mM glycylglycine, 26.4mM NMG, ImM EGTA, to produce buffers of differing pH value as re- quired. The cheek cell suspension was acidified at room temperature for 3 hours in approximately 20 volumes of pH 5.5 buffer (for proton-dependent and non proton-dependent 22 Na + uptake) or in pH 7.8 buffer (for non proton-dependent 22 Na + uptake) . Samples were centrifuged as described and resuspended in their respective buffers to give a final protein concentration of between 5 and 15 mg/ml plus or minus the addition of ImM ouabain, ImM bumetanide, or ImM amiloride and further incubated at 25°C for 15 minutes. The timed proton-dependent reaction was initiated at 25°C by a ten-fold dilution of acidified (pH 5.5) cells into uptake buffer normally at pH 7.8 which gave a final pH in the assay of 7.5 and hence a transmembrane proton gradient (inside → outside) of 100:1. Measurements were also made using uptake buffers of differing pH to produce a range of final proton gradients as indicated. Non proton-dependent sodium uptake was measured by adding cells loaded at pH 5.5 or pH 7.8 to buffer of the same pH. Uptake buffer for the 100:1 proton gradient contained a final concentration of 230mM mannitol, 20.8mM NMG, 18mM MES, 10.7mM glycylglycine, ImM EGTA, ImM Na + (gluconate) and 22 Na + (Cl) (carrier free) at 2xl0 7 cpm/ml (6.4uCi/ml), 500-1500ug/ml cheek cell protein plus or minus ImM ouabain. Na + uptake was also measured in the presence of ImM amiloride. The reaction was terminated at various times as indicated by adding 25ul aliquots of the reaction mix to 4 ml ice-cold wash buffer containing ImM NaCl, lOOmM mannitol, lOOmM MgCl 2 , 8mM HEPES, 4mM TRIS, pH 7.2. Cells containing 22 Na + were collected by rapid filtration through 0.45 micron Millipore filters which were then washed twice with 4 ml ice-cold wash buffer. Filters were dried and covered with 4mls Dupont Econofluor scintillant and counted in a Wallac 1410 beta-counter. Measurements were made in duplicate or triplicate and background 22 Na + retained on filters in the absence of cheek cells was subtracted from the assay values. Cheek cell protein was determined by the method of Lowry et al., (1951) and controlled for the background colour reading produced by NMG. Rat kidney brush border membrane vesicles (BBMV) exhibiting high levels of proton-dependent Na + uptake were prepared according to the method of Hilden et al., (1989) using Mg 2 * aggregation with sequential centrifugation. Na + /H + antiporter activity of BBMV was measured with every daily set of cheek cell assays as described above as a check on the assay procedure. Cell counting and vital staining Small aliquots of cells from the final pH 5.5 cell suspension were diluted in a congo red or trypan blue solution to give a final stain concentration of 0.06% (w/v). Cells were examined under an Olympus microscope at x400 magnification using an Improved Neubauer haemocytometer. The cells were counted and inspected for their inclusion or exclusion of vital stain. Chemicals AmilorideΗCL, bumetamde and ouabain were obtained from Sigma Chemical Co. All other reagents were of the highest commercial grade available. Appendix 2 Materials and methods for measuring sodium transport in the adult cheek cell study. Subjects Normotensive and hypertensive subjects were recruited from a total of 1100 adult male and female volunteers who participated in a blood pressure screening program. The hypertensive group comprised subjects having an average of their second and third sitting diastolic blood pressure readings of ≥95mm Hg when measured with a Dinamap automatic blood pressure recorder and were aged between 25 and 60 years. The control group had a diastolic blood pressure of <85mm Hg and were matched to the hypertensive group for age and gender. None of the control or hypertensive subjects selected for the final study were receiving antihypertensive medication at the time of the blood pressure screening or during the collection of cheek cells. On the same morning of cheek cell sampling, the blood pressure was re-measured to confirm previous readings. Characteristics of the respective study groups are shown in Table 1. Collection of cheek cells Cheek (buccal mucosal) cells were obtained in a non-invasive manner by having subjects swish distilled water around their mouth for a short period of time, using a gentle molar scraping action. The expectorate containing the cheek cells was collected and a further two or three 10ml washes produced an average total yield of about 4 million cells. Samples were spun at 46,000g x 15 minutes in a Beckman J2-21 centrifuge using a JA-20 rotor at 4°C. The packed cheek cell pellet was resuspended in 1ml distilled water by gentle homogenisation. Cheek cells were isolated from subjects in the morning and assayed for Na + /H + antipo¬ rter activity on the same day. There was no significant difference between the cell yield of the normotensive group compared to the hypertensive group. Measurement of Na + uptake by the Na + /H + antiporter assay Na + /H + antiporter activity was measured by the uptake of 22 Na + using a modifi¬ cation of the Millipore filtration technique as described by Seiler et al.,(1985). 700ul of the cheek cell suspension was acidified at room temperature for 3 hours in 15ml of Buffer I (pH 5.5) containing 230 mM mannitol, 49mM MES, 11.2mM N-methyl-D-glucamine (NMG), and ImM ethyleneglycol-bis-(j8-amino-ethyl ether)N,N'-tetra-acetic acid (EGTA) (for proton-dependent and non proton-depe¬ ndent 22 Na + uptake). The remainder (300ul) was equilibrated in 1.5ml of Buffer π (pH 7.8) containing 230mM mannitol, 33.6mM HEPES, 26.4mM NMG and ImM EGTA (for non proton-dependent 22 Na + uptake). Samples were centrifuged as described above, and resuspended in their respective buffer plus the addition of ImM ouabain, and further incubated at 25°C for 15 mins. The timed proton-dependent reaction was imtiated at 25°C by a ten-fold dilution of pH 5.5 loaded cells (15ul) into uptake buffer (150ul final volume) consisting of Buffer H titrated with MES to pH values of 7.05, 7.35, 7.65 and 7.8. This provided final proton gradients (inside → outside) of 19:1, 40:1, 75:1 and 100:1, respectively. Non proton dependent 22 Na + uptake was measured by adding cells loaded at pH 5.5 or pH 7.8 to uptake buffer of the same pH. For the 100:1 proton gradient, the reaction buffer contained a final concentration of 230mM mannitol, 4.9mM MES, 30.2mM Hepes, 24.9mM NMG, ImM EGTA, ImM Na + (gluconate) and 22 Na + (Cl) (carrier free) at 13.3xl0 6 cpm/ml (4.3uCi/ml), 500-1500 ug/ml cheek cell protein, plus or minus ImM ouabain, pH 7.5. Na + uptake was also measured in the presence of ImM bumetamde. The reaction was terminated at 30 seconds and 5 minutes by adding 25ul aliquots of the reaction mix to 4 ml ice-cold wash buffer containing ImM NaCl, lOOmM mannitol, lOOmM MgCl 2 , 8mM HEPES, 4mM TRIS, pH 7.2. Cells containing 22 Na + were collected by rapid filtration through 0.45 micron Millipore filters which were then washed twice with 4ml ice-cold wash buffer. Measurements were made in duplicate (30 seconds) and triplicate (5 min). Filters were dried and covered with 4ml DuPont Econofluor scintillant and counted in a Wallac 1410 beta-counter. Background values for 22 Na + retained on the filters in the absence of cheek cells were subtracted from the assay values. Cheek cell protein was determined by the method of Lowry et al., (1951) and controlled for the background reading produced by NMG. As a control for variability, weekly checks of cheek cell Na + /H + antiporter activity of cells from a small number of normotensive volunteers were made throughout the duration of the study. In addition, rat kidney brush border membrane vesicles exhibiting high levels of proton-dependent sodium uptake (prepared as described by Hilden et al., 1989) using Mg 2+ aggregation with sequential centrifugation, were included with every daily set of assays as a check on the assay procedure. Cheek cell counting and trypan blue staining Small aliquots of cells were sampled from the final cell preparations at pH 5.5 and pH 7.8 and diluted ten-fold in distilled water. An aliquot of this dilution was added 1:1 with a neutral filtered aqueous 0.2% (w/v) trypan blue solution to give a final stain concentration of 0.1 % (w/v). Cells were examined under an Olympus microscope at x400 magnification using an Improved Neubauer haemacytometer that gave a viewing depth of 200um. The cells were counted and inspected for their inclusion or exclusion of trypan blue. Chemicals Bumetanide and ouabain were obtained from Sigma Chemical Co. Sodium-22 was from New England Nuclear. All other reagents were of the highest commercial grade available. Appendix 3 Materials and methods for measuring sodium transport in the adolescent cheek cell study. Subjects Subjects were recruited from a 3-year blood pressure tracking study. Systolic and diastolic blood pressures were measured for 504 male and female high school students from two schools in 1986/87 and again in 1989/90. The setting up and actual running of these adolescent blood pressure surveys were carried out by Dr PRC Howe, CSIRO, Division of Human Nutrition, Adelaide, to whom we are most grateful. For both occasions, systolic blood pressure readings were re¬ gressed on age and age squared. Weight, height and obesity were not taken into account. Deviations of each student's observation from the expected regression line were calculated, and from this each student was assigned a rank. The low BP and high BP tracking groups were identified from the upper and lower eight percent of the ranked sum of the two systolic blood pressure readings. Informed consent was obtained from 35 high and 32 low tracking adolescents. A final set of blood pressure readings in the seated position were taken using a Dinamap automatic blood pressure recorder on the day cheek cells were collected and sodium transport assayed. Characteristics of the study group are shown in Table 3. Six subjects from the high BP tracking group and eight subjects from the low BP tracking group recorded a final systolic blood pressure (on the day of cheek cell sampling) which was less than or greater than one standard deviation from the mean of their respective group. These subjects were not included in the final analyses. Collection of cheek cells Cheek (buccal mucosal) cells were obtained in a non-invasive manner by having subjects swish distilled water around their mouth for a short period of time, using a gentle molar scraping action. The expectorate containing the cheek cells was collected and a further two or three 10ml washes produced an average total yield of about 4 million cells. Samples were spun at 46,000g x 15 minutes in a Beckman J2-21 centrifuge using a JA-20 rotor at 4°C. The packed cheek cell pellet was resuspended by gentle homogenisation in loading buffer (pH 5.5) as described below. Cheek cells were isolated from subjects in the morning and assayed for Na + /H + antiporter activity on the same day. Measurement of Na + transport by the Na + /H + antiporter assay Na + /H + antiporter activity was measured by the uptake of 2 Na + using a modifi- cation of the Millipore filtration technique as described by Seiler et al., (1985). All cell equilibration and transport buffers used were prepared by titrating a pH 5.5 buffer: 230mM mannitol, 49mM MES, 11.2mM N-methyl-D-glucamine (NMG), ImM ethyleneglycol-bis-(j8-amino-ethyl ether)N,N'-tetra-acetic acid (EGTA), with a pH 8.4 buffer: 230mM mannitol, 16.8mM glycylglycine, 26.4mM NMG, ImM EGTA to produce buffers of differing pH value as re¬ quired. Cheek cells were acidified at room temperature for 3 hours in approximately 20 volumes of pH 5.5 loading buffer (for proton-dependent and non proton-depend- ent 22 Na + uptake) or in pH 7.8 buffer (for non proton-dependent 22 Na + uptake). Samples were centrifuged as described above, and resuspended in their respective buffers to give a final protein concentration between 5 and 15 mg/ml plus or minus the addition of ImM ouabain and/or ImM amiloride and further incubated at 25°C for 15 mins. The timed proton-dependent reaction was initiated at 25°C by a ten-fold dilution of acidified (pH 5.5) cells into uptake buffer (150ul final volume) normally at pH 7.8 to give a final assay pH of 7.5 and hence a transmembrane proton gradient (inside → outside) of 100:1. Measurements were also made using uptake media of differing pH to produce a range of final proton gradients as indicated. Non proton-dependent sodium uptake was measured by adding cells loaded at pH 5.5 to buffer of the same pH. Uptake buffer for the 100:1 proton gradient contained a final concentration of 230mM mannitol, 20.8mM NMG, 18mM MES, 10.7mM glycylglycine, ImM EGTA, ImM Na + (gluconate) and 2 Na + (Cl") (carrier free) at 2xl0 7 cpm/ml (6.4 Ci/ml), 500-1500ug/ml cheek cell protein, plus or minus ImM ouabain. Na + uptake was also measured in the presence of ImM amiloride. The reaction was terminated at various times as indicated by adding 25ul aliquots of the reaction mix to 4ml ice-cold wash buffer containing ImM NaCl, lOOmM mannitol, lOOmM MgCl 2 , 8 mM HEPES, 4mM TRIS, pH 7.2. Cells containing - ~ N& + were collected by rapid filtration through 0.45 micron Millipore filters which were then washed twice with 4ml ice-cold wash buffer. All measurements were made in duplicate (for 30 seconds and 10 minutes) or triplicate (for 2 and 5 minutes). Filters were dried and covered with 4ml Dupont Econofluor scintillant and counted in a Wallac 1410 beta-counter. Background values for ^Na* retained on the filters in the absence of cheek cells were subtracted from the assay values. Cheek cell protein was determined by the method of Lowry et al., (1951) and controlled for the background colour reading produced by NMG. As a control for variability, weekly checks of cheek cell Na + /H + antiporter activity from a small number of normotensive volunteers were made throughout the duration of the study. In addition, rat kidney brush border membrane vesicles exhibiting high levels of proton-dependent sodium uptake (prepared as described by Hilden et al., 1989) using Mg 2 * aggregation with sequential centrifugation, were included with every daily set of assays as a check on the assay procedure. Chemicals AmilorideΗCL and ouabain were obtained from Sigma Chemical Co. Sodium-22 was from New England Nuclear (Australia). Appendix 4 Statistics All data are presented as the mean ± the standard error of the mean with the significance of differences between means for each assay condition/time point for the study groups, being determined by Student's unpaired t-test. Appendix 5 UMMARY F N* TRANSPORTUPTAKE ACTIVITY Pf VARIOUS TIS UE 1 BBMV - Brush border membrane vesicles 2 WKY - istar-Kyoto rat 3 SHR - Spontaneously hypertensive rat Appendix 6 TABLE 3 COMPARISON OF PREVIOUS STUDIES ON CATION TRANSPORT IN RED BLOOD CELLS, LEUCOCYTES AND PLATELETS OF HYPERTENSIVE AND NORMOTENSIVE SUBJECTS WITH THE PRESENT CHEEK CELL MARKER FOR HUMAN HYPERTENSION STUDY 'Hn Nn Ηx/Nx(%) 4 N>Hx(%H) 5 H<Nx(%H) 'OVERLAP Li*-Na* Countertransport- red blood cells Weder 1986 <0.05 Weinberger et al 1989 <0.01 Weinberger et al 1989 <0.01 Carr et al 1990 <0.01 3/23(13%) 3/13(23%) 18% Yap et al 1989 N.S. Morgan et al 1988 not given Canessa et al 1980 <0.001 0/36(0%) 0/36(0%) 0% Semplicini et al 1989* <0.05 4/21(19%) 14/41(34%) 26% Adragna et al 1982 <0.001 Woods et al 1982 <0.001 Canali et al 1981 <0.001 Cusi et al 1981 <0.01 Trevisan et al 1983 <0.05 Williams et al 1983 <0.001 Clegg et al 1982 <0.001 Weder et al 1984 <0.05 Wiley et al 1984 N.S. Na*-H* Countertransport - Red blood cell Morgan et al 1988 169% <0.005 Semplicini et al 1989* 172% <0.01 2/21(9%) 11/41(27%) 18% Na + -H + Countertransport- leuco Ng et al l989 141% <0.05 0/17(0%) 4/17(23%) 11.5% Ng et al 1990 154% <0.001 Wehling et al 1991 104% <0.05 4/24(17%) 2/12(17%) 17% Na*-H* Countertransport - platelets Livne et al 1987 7 20 195% <0.005 «N<Hx(%N) 7 H>Nx(%H) Cheek Cell proton-dependent Na* uptake (This study) Proton gradient (75:1) 15 19 34% <0.0004 1/19(5%) 1/15(7%) 6% [Nx/Hx(%) 293%] Slope with proton 15 19 4% <0.003 2/19(10%) 2/15(13%) 11.5% gradient (19:1-75:1) [Nx/Hx(%) 2400%] *The study by Semplicini et al 1989 had as its principal investigator M. Canessa and was a follow up study of the one done by Canessa et al 1980. 'Hn and Nn refers to the number of hypertensives (H) and normotensives (N) in each study. 2 Hx/Nx(%) refers to the percentage increase (or decrease) between the mean rate of cation transport in the system under investigation in hypertensives (Hx) and normotensives (Nx). 'Probability that differences between groups were significant as determined by the statistical treatment used in each study. N.S., not significant. Number (%) of normotensives having a value greater than the mean of the hypertensive group. ^Number (%) of hypertensives having a value less than that the mean of the normotensive group. 'Number (%) of normotensives having a value less than that the mean of the hypertensive group. 'Number (%) of hypertensives having a value greater than the mean of the normotensive group. 'Average of (4) and (5) or (6) and (7). REFERENCES Adragna N.C., Canessa M ., Solomon H., Slater E. and Tosteson D.C. (1982) Red cell Li + /Na + exchange in patients with essential hypertension. 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Claims: CLAIMS 1. A method for the determination of a physiological abnormality in a human or animal subject, which method includes determining ion flux across the membrane of epithelial cells taken from said subject. 2. The method of claim 1 wherein the epithelial cell is selected from a cheek epithelial (buccal mucosal) cell, a skin dermal epithelial cell, a bladder epithelial cell, a nasal epithelial cell or the like. 3. The method of claim 2 wherein the epithelial cell is a cheek epithelial (buccal mucosal) cell. 4. The method of claim 1 wherein the ion is a cation. 5. The method of claim 4 wherein the ion is sodium ion (Na + ). 6. The method of claim 4 wherein the ion is a proton (H + ) 7. The method of claim 1 wherein the subject is human and the physiological abnormality is hypertension or pre-disposition towards hypertension. 8. An assay suitable for use in the method of any one of claims 1 to 7 said assay comprising determining the flux of a first ion across the membrane of an epithelial cell under an imposed concentration gradient of a second ion. 9. The assay of claim 8 wherein the first and second ions are cations. 10. The assay of claim 8 wherein the first ion is a sodium ion (Na + ) and the second ion a proton (H + ). 11. The assay of claim 8 wherein the epithelial cell is selected from a cheek epithelial (buccal mucosal) cell, a skin dermal epithelial cell, a bladder epithelial cell, a nasal epithelial cell or the like. 12. The assay of claim 11 wherein the epithelial cell is a cheek epithelial (buccal mucosal) cell. 13. The assay of claim 10 wherein the proton (H + ) concentration gradient across the cell membrane (inside → outside) is in the range of about 10:1 to about 100:1. 14. The assay of claim 13 wherein the proton (H + ) concentration gradient is about 75:1. 15. The assay of claim 13 wherein the determination is carried out at two or more proton concentration gradients. 16. The assay of claim 8 wherein the cell is exposed to a medium containing a Na + concentration in the range 50 micromole to 150 millimole per litre. 17. The assay of claim 16 wherein the Na + concentration is about 1 millimole per litre. 18. The assay of claim 8 wherein the determination is carried out in the presence of an (Na + + K + )ATPase inhibitor. 19. The assay of claim 18 wherein the inhibitor is ouabain. 20. The assay of claim 8 wherein the ion flux is determined over a period ranging from about 15 seconds to about 40 minutes incubation at about 25°C. 21. The assay of claim 20 wherein the ion flux is determined over a period less than 10 minutes. 22. The assay of claim 10 wherein Na + flux is measured by determining cell uptake of radioactively labelled Na + 23. The assay of claim 10 wherein the Na + flux is determined by measuring H + movement across the cell membrane. 24. The assay of claim 10 where Na + or H + are measured using an ion specific fluorescent probe. 25. A method for the determination of hypertension and/or pre-disposition towards hypertension in a human subject, said method comprising the steps of: establishing a proton (H + ) concentration gradient across the membrane of epithelial cells taken from said subject, the proton concentration being greater inside said cells than outside said cells; and determining the proton-dependent uptake of sodium ions (Na + ) by said cells when said cells are exposed to medium containing sodium ion (Na + ). 26. The method of claim 25 wherein the epithelial cell is selected from a cheek epithelial (buccal mucosal) cell, a skin dermal epithelial cell, a bladder epithelial cell, a nasal epithelial cell or the like. 27. The method of claim 26 wherein the epithelial cell is a cheek epithelial (buccal mucosal) cell. 28. The method of claim 25 wherein the proton (H + ) concentration gradient across the cell membrane (inside -» outside) is in the range of about 10:1 to about 100:1. 29. The method of claim 28 wherein the proton concentration gradient is about 75:1. 30. The method of claim 28 wherein the determination is carried out at two or more proton concentration gradients. 31. The method of claim 25 wherein the cell is exposed to a medium containing a Na + concentration in the range 50 micromole to 150 millimole per litre. 32. The method of claim 31 wherein the Na + concentration is about 1 millimole per litre. 33. The method of claim 25 wherein the determination is carried out in the presence of an (Na + + K + )ATPase inhibitor. 34. The method of claim 33 wherein the inhibitor is ouabain. 35. The method of claim 25 wherein the Na + uptake is determined over a period ranging from about 15 seconds to about 40 minutes incubation at about 25°C. 36. The method of claim 35 wherein the Na + uptake is determined over a period less than 10 minutes. 37. The method of claim 25 wherein Na + uptake is measured by determining cell uptake of radioactively labelled Na + . 39. The method of claim 25 wherein the Na + uptake is determined by measuring H + movement across the cell membrane. 40. The method of claim 25 where Na + and/or H + are measured using an ion specific fluorescent probe. 41. The method of claim 25 when used for the determination of hypertension in an adult. 42. The method of claim 25 when used for the determination of a predisposition to hypertension in a subject. 43. The method of claim 25 which comprises the steps of: (i) incubating a sample of epithelial cells taken from the subject in an acid medium; (ϋ) contacting the acidified cells with a less acid medium containing sodium ions (Na + ); (iii) separating the cells from the less acid medium; and (iv) determining the uptake of sodium ions (Na + ) by the cells. 44. An assay t for use in the assay of claim 8 or the method of claim 25, said kit including an acid medium in a suitable container for acidifying epithelial cells and a second less acid medium containing sodium ions (Na + ) in a suitable container. 45. An assay kit according to claim 44 said kit including an indicator for detection of ion concentration in a suitable container, and an inhibitor of (Na + +K + )ATPase activity in a suitable container. 46. The kit of claim 45 wherein the inhibitor is ouabain. 47. The kit of claim 46 wherein the indicator is a fluorescent probe for Na + and/or H + . 48. The kit of claim 47 further including a predetermined mix of organic phthalates in a suitable container. 49. An assay t for use in the assay of claim 8 or the method of claim 25, said Mt including radioactive Na + in a suitable container, and an inhibitor of (Na + +K + )ATPase activity in a suitable container. 50. The Mt of claim 49 wherein the inhibitor is ouabain. 51. The Mt of claim 49 further including a predetermined mix of organic phthalates in a suitable container.

Inventors: MCMURCHIE EDWARD JOHN, MCMURCHIE, EDWARD, JOHN
Applicants: COMMW SCIENT IND RES ORG, COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH OR
Application year: 1991
Main classification: C12Q1
IPC codes: C12Q1, G01N33