WO1999024079A1

WO1999024079A1 - Improved labelled diagnostic compositions and methods of their use

Info

Publication number: WO1999024079A1
Application number: PCT/AU1998/000939
Authority: WO
Inventors: Trevor G. Redgrave; Ian J. Martins
Original assignee: Peptide Delivery Systems Pty. Ltd.
Priority date: 1997-11-11
Filing date: 1998-11-11
Publication date: 1999-05-20
Also published as: AUPP032097A0

Abstract

This invention relates to a non-radioactively labelled diagnostic composition for testing the presence of or propensity for atherosclerosis and coronary artery disease and the like comprising components which mimic essential features of an exogenous lipoprotein transport particle, said composition being capable of metabolisation by normal physiological pathways such that at least one non-radioactively labelled metabolite is detectable in the blood or bodily waste of a patient being tested. Further, it also relates to a method of determining the presence of or propensity for atherosclerosis and coronary artery disease and the like in a patient requiring such determination comprising administering to said patient a non-radioactively labelled diagnostic composition which mimics essential features of an exogenous lipoprotein transport particle, and measuring the quantity of labelled metabolite in the bodily waste or blood of said patient so as to determine the quantity of labelled diagnostic composition successfully metabolised by said patient.

Description

IMPROVED LABELLED DIAGNOSTIC COMPOSITIONS AND METHODS OF THEIR USE

FIELD OF THE INVENTION

This invention relates to non-radioactive formulations useful in the diagnosis of a patient's predisposition to the development of atherosclerosis and coronary artery disease. It also relates to a method for producing such formulations and to a method of diagnosing a patient's tendency to develop the aforementioned diseases.

BACKGROUND ART

The predisposition of a person to develop atherosclerosis is dependent on several factors including genetic inheritance and environment. Atherosclerosis is a progressive disease of the walls of blood vessels, beginning with the accumulation of lipid substances and over a period of years leading to pathological changes such as fibrosis, ulceration and thrombosis within the vessel wall.

Serious diseases consequent upon atherosclerosis include myocardial infarction, aneurism, stroke and failure of adequate blood supply to organs and limbs leading to organ failure and gangrene. The disease is very common in Western countries where a diet high in cholesterol, excessive fat ingestion, cigarette smoking, obesity and physical inactivity are all contributing factors. Genetic factors can also lead to the onset of atherosclerosis.

Although methods such as angiography and measurements of blood flow currently exist for assessment of established atherosclerosis, there are no current, accurate tests available to determine the likelihood that a patient has a metabolic predisposition towards development of the disease. Since treatments are available and preventative behavioural changes possible, such a method of predetermining a propensity to develop atherosclerosis would be useful to both patient and physician.

Chylomicrons transport fat in the form of t acylglycerols from the small intestine to fat depots. The t glycerides in these particles are subject to a hydrolysis action by lipoprotein upases in the bloodstream resulting in formation of a secondary particle which still contains up to 30% thglycerides, and the whole of the particle cholesteryl ester. This secondary particle is known as a chylomicron remnant or chyloremnant. The chyloremnant, having been divested of much of its triglyceride content, then contains a much higher relative amount of cholesterol to phospholipid. The primary function of the chyloremnant is to transport cholesterol from the intestine to the liver.

It has been found that an individual's ability to metabolise chyloremnants may be directly related to their propensity to develop atherosclerosis, and thus measurement of chyloremnants, or their precursor particles, or chylomicrons is desirable as a diagnostic tool for atherosclerosis and related diseases. A reduced tolerance for chyloremnant clearance would indicate increased risk of atherosclerosis, while repeated tolerance tests would be useful in judging the effectiveness of therapies and interventions aimed at reducing atherosclerosis risk.

With the above in mind, applicant's international application PCT/AU 94/00663 teaches radioactively labelled compositions and methods for diagnosing a patients predisposition to atherosclerosis and coronary heart disease. Whilst effective, radioactively labelled compositions suffer from a number of drawbacks. Radioactivity adds to risk of cancer and of genetic mutations. This risk will be confined to the individual recipient if isolated from the environment. However exhaled radioactivity from an unconfined recipient will contaminate the external environment and add to risks of cancer and genetic mutations in laboratory staff and family or others in contact with the recipient. Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

DISCLOSURE OF THE INVENTION

The present invention provides a non-radioactively labelled diagnostic composition for testing for the presence of or propensity for atherosclerosis and coronary artery disease and the like comprising components which mimic essential features of an exogenous lipoprotein transport particle, said composition being capable of metabolisation by normal physiological pathways such that at least one non-radioactively labelled metabolite is detectable in the blood or bodily waste of a patient being tested.

Preferably, the exogenous lipoprotein transport particle is one which mimics the chemical formulation of a chyloremnant, ie, it is preferably one which is primarily lipidic in nature.

Whilst non-radioactively labelled compositions have their advantages, producing such compositions is not a straight forward task. In particular, non-radioactively labelled compositions must be capable of being administered in a way that utilises the physiological pathway of interest, without producing the phenomenon of saturation, yet in sufficient amount to allow accurate detection by measurement techniques that are generally available. The present invention solves this problem by providing artificial lipoprotein emulsion particles enriched with cholesteryl esters and the like, labelled in the fatty acid moiety with a non- radioactive carbon label. Although any fatty acid moiety may be labelled, preferably the non-radioactive carbon label is located within the cholesteryl ester component of the composition. It will be appreciated however, that compounds other than cholesteryl esters may be used provided that they behave as do cholesteryl esters. Suitable compounds may include vitamin A derivatives and non-endogenous xenobiotics. In a preferred form of the invention, the label is attached to the fatty acid moiety of the cholesteryl ester. Other chemistries of attaching the label to the cholesteryl ester are also possible.

To avoid insensitivity when testing for atherosclerosis and coronary artery disease and instability of exogenous lipoprotein transport particles the cholesteryl ester must be present in amounts of between about 10 to 40% of the total mass of lipid. Preferably, the cholesteryl ester is present in amounts of between about 10 to 30% of the total mass of lipid. More preferably, the cholesteryl ester is present in amounts of between about 15 to 25% of the total mass of lipid. In a highly preferred example of the invention the cholesteryl ester is present in amounts of between about 16.5 to 22.5% of the total mass of lipid.

Therefore, in a preferred form, the invention provides a non-radioactively labelled diagnostic composition for testing the presence of or propensity for atherosclerosis and coronary artery disease and the like, said composition comprising:

(i) a plurality of lipidic components selected in such a manner so as to mimic the essential features of a chyloremnant wherein at least one of the components is cholesteryl ester that is present in an amount of between 16 to 23% of the total mass of lipid; and wherein the composition is capable of metabolisation by normal physiological pathways such that at least one non-radioactively labelled metabolite is detectable in the blood or bodily waste of a patient being tested.

Preferably, the non-radioactive label is one that allows for the measurement of the amount of metabolite present by mass spectrometry. For example, ¹³C may be used so that the isotope enrichment ratio of ¹³C relative to ¹²C in the CO₂ of the expired breath of a patient can be measured. Other equivalent, physiologically tolerated, stable, non-radioactive isotopes or other labels could also be incorporated into the composition. Size is a determinant of metabolism, and to this extent, it is preferred that the particles be of a size that is easily metabolised. In particular, it is preferred that the non-radioactively labelled diagnostic composition comprises particles of a diameter from 10-1000 nanometers (nm). Surprisingly, size has also been found to be an important criterion for sterilisation (for example by filtration) and stability of the particles during freezing. Preferably, the size of the particles in the emulsion is kept as small as possible. Emulsions are generally regarded as unstable during freezing because of crystallisation of the constituents leading to particle instability. However, mixtures of lipids in sufficiently small particles when in solutions containing glycerol were able to be supercooled or frozen and thawed without instability of the emulsified particles. Most preferably, the size of the particles is between about 10-200nm. Desirably the range is between about 20-60nm, while particles sizes between about 30 to 40nm are highly preferred. For example the particles sizes should be approximately 35nm.

Therefore, in a more highly preferred form, the invention provides a non- radioactively labelled diagnostic composition for testing the presence of or propensity for atherosclerosis and coronary artery disease and the like, said composition comprising:

(i) a plurality of lipidic components selected in such a manner so as to mimic the essential features of a chyloremnant wherein at least one of the components is cholesteryl ester that is present in an amount of between 16 to 23% of the total mass of lipid; and

(ii) the particles have an average size of approximately 35 nm; wherein the composition is capable of metabolisation by normal physiological pathways such that at least one non-radioactively labelled metabolite is detectable in the blood or bodily waste of a patient being tested.

The composition used in the invention should preferably be one that results in no untoward local or systemic reaction following introduction to the patient being tested, and should, preferably be well tolerated by the patient upon repeated administration. Preferably, the composition according to the invention includes triglycerides, phospholipids, cholesterol, and cholesteryl esters, at least one of these having a suitable located label.

Cholesteryl esters which are suitable for use in the composition of the invention include cholesteryl oleate, cholesteryl linoleate or a mixture of related esters. Cholesteryl esters are generally the carrier of the label which enables measurement of the metabolisation of the compositions according to the invention. The fatty acid moiety of the cholesteryl ester is preferably that labelled.

Thglycerides, which may be present in the composition in an amount of 20-95% of total lipids, and which are suitable for use in the composition include triolein (TO), soybean oil, safflower oil, or other suitable substitutes such as olive oil or corn oil thglycerides.

Suitable phospholipids include egg yolk phosphatidylcholine (PC), soybean phosphatidylcholine, dioleoyl phosphatidylcholine, or other suitable substitutes. This lipid may be present in an amount of 5-50% of total lipids.

Cholesterol which should be present in amounts adequate for the metabolism of the composition to mimic that of the natural exogenous lipid transport particles, is preferably present in amounts of 5-20% of total lipids. Without cholesterol it has been found that the chyloremnants remain in the plasma for much longer than occurs physiologically, and thus a composition not having cholesterol is not able to properly mimic the natural exogenous lipid transport particles and is therefore not entirely suitable for the purposes of the invention. Preferably cholesterol is present in an amount of about 5 to 15% total lipid, while 7 to 10% appears optimal. If the amount of cholesterol is too high the particles become unstable and are phagocytosed by the reticulo-endothelial system when introduced into a patient. If the amount of cholesterol is too low the particles are not taken up by hepatocytes. ln a preferred diagnostic composition according to the invention there is present 16 to 23% cholesteryl ester, 30-60% thglycerides, 8-30% phospholipids and about 7 to 10% cholesterol.

In a highly preferred composition according to the invention, there is present approximately 46.5% triolein, 16.5% cholesteryl oleate, 9.5% cholesterol and 27.5% egg phosphatidylcholine.

The components of the composition, including the labelled component are preferably emulsified in a solution in water of glycerol or glycerol and salts, for example sodium chloride and buffer salts. The presence of the glycerol improves the stability in storage of the composition and makes it isotonic. The composition may additionally include a small quantity of an anti-oxidant to preserve the lipids against oxidation. Such anti-oxidants as ascorbic acid or vitamin E are suitable. To stably emulsify the composition, well known techniques such as sonication or microfluidization may be used. Emulsion particle size affects the kinetics of clearance, so it is desirable that the emulsion preparation procedures result in particles of the preferred size as set out hereinabove.

In total, the lipid content of the composition is preferably present in amounts of 1- 50% of the total aqueous-based emulsion.

For ease and accuracy of quantitative measurement of the patient's propensity to develop atherosclerosis, the labelled metabolite may be measured in the captured expired air of the patient, although a patients propensity for atherosclerosis may also be measured from samples of urine, or blood.

Preferably, the composition is in the form of an emulsion for intravenous injection although other administration routes may be possible. If, for example, the composition is to be administered orally, it should be suitably formulated so as to be resistant to gastric degradation. The present invention also provides a method of determining the presence of or propensity for atherosclerosis and coronary artery disease and the like in a patient requiring such determination comprising administering to said patient a labelled diagnostic composition which mimics essential features of an exogenous lipoprotein transport particle, and measuring the quantity of labelled metabolite in the bodily waste or blood of said patient so as to determine the quantity of labelled diagnostic composition successfully metabolised by said patient.

Although this method may be used to ascertain the level of development of atherosclerosis by a patient, because chyloremnant metabolism may vary from person to person, the method is also useful for gauging over an interval of repeated tests, a patient's decline towards an atherosclerosis state.

Most desirably, the composition mimicking the essential features of the exogenous lipoprotein particle is delivered to the patient by injection.

In a preferred embodiment of this aspect of the invention said labelled diagnostic composition is one which mimics the chemical formulation of a chyloremnant, that is, it is primarily lipidic in nature.

For optimum analysis, the measurement of the quantity of labelled metabolite present in the blood or bodily waste of the individual is measured from the patient's expired air, and is preferably carried out at intervals over a period of several hours. Measurements of the quantity of labelled metabolite may commence as early as 15 minutes after inoculation of the patient with the compositions according to the invention, and may be concluded as long as six hours or more after the initial administration of the composition to the patient.

The individuals expired air may be collected in a known fashion by trapping the expelled air in a vessel containing solutions which trap CO₂ created as a result of the individuals metabolism. Whilst the labelled diagnostic composition is preferably administered as an emulsion by intravenous injection, it may also be administered by a suitable alternative route such as orally.

A preferred composition administered in accordance with this aspect of the invention includes 20-95% by weight of total lipids of triglycerides, 5-50% by weight of total lipids of phospholipids, 2-20% by weight of total lipids of cholesterol, and 10-40% by weight of total lipids of cholesteryl esters, at least a small proportion of which may be labelled.

Suitable cholesteryl esters, triglycerides, phospholipids and sources of cholesterol are as previously described.

In one preferred method according to the invention, the labelled diagnostic composition comprises 16-23% cholesteryl ester, 30-60% triglycerides, 8-30% phospholipids and 7-10% cholesterol.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in the following non-limiting Figures and Examples.

Figure 1 shows a graph showing the enrichment of ¹³CO₂ in the expired breath of control and apo E knockout mice.

Figure 2 shows that abnormal remnant metabolism forms part of the metabolic syndrome accompanying visceral obesity. BEST MODE(S) FOR CARRYING OUT THE INVENTION

Examples

Further features of the present invention are more fully described in the following Examples. It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the invention, and should not be understood in any way as a restriction on the broad description as set out above.

Example 1 Preparation of chyloremnants for use In rats

Lipid mixtures containing TO (70 mg), PC (25 mg), cholesteryl [¹ ^C]-oleate (3 mg), and cholesterol (2 mg) were emulsified by sonication for 1 h in 8.5 ml of 2.2

% glycerol in water. Uniformly labelled [¹³C]-oleic acid was purchased from

Novachem Pty. Ltd., Victoria, Australia, and cholesteryl [^C]-oleate was synthesised from cholesterol and [¹³C]-oleic acid as described previously (Mortimer, B-C. et al., (1990) "The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats." Biochem. Biophvs. Acta. 1046, 46-56). After sonication, the emulsion mixture was centrifuged at 3000 rpm for 10 min to remove titanium fragments and then filtered through a 0.22μm filter into sterile vessels.

Example 2 Testing chyloremnant particles in mice

A volume of 100 μl of the emulsion mixture produced in Example 1 was injected via a tail vein into C57BL/6J (control) mice and apo E knockout mice. Colonies of apo E "knockout" mice were established from progenitor stocks obtained from the Jackson Laboratories, (Bar Harbor, ME). The mice were placed in a closed chamber through which a stream of room air was passed as previously described (Redgrave, T.G., et al., (1995). "Measurement of expired carbon dioxide to assess the metabolism of remnant lipoproteins." J. Lipid Res. 36, 2670-2675). Breath samples were collected at intervals into evacuated gas sample containers (Europa Scientific Ltd, Crewe, U.K.). The enrichment of breath samples with ¹³CO₂ was measured by isotope ratio-mass spectrometry (ABCA, Europa Scientific, Crewe, U. K.). The results are shown in Figure 1.

Example 3 Preparation of chyloremnants for use in humans

Lipid mixtures containing TO (135 mg), PC (75 mg), cholesteryl [^{1 3}C]-oleate (69 mg), and cholesterol (24 mg) were emulsified by sonication for 1 h in 8.5 ml of 2.2% glycerol in water. Uniformly labelled [¹³C]-oleic acid was purchased from

Novachem Pty. Ltd., Victoria, Australia, and cholesteryl [^Cj-oleate was synthesised from cholesterol and [¹³C]-oleic acid as described previously (Mortimer, B-C. et al., (1990) "The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats." Biochem. Biophvs. Acta. 1046, 46-56). After sonication, the emulsion mixture was centrifuged at 3000 rpm for 10 min to remove titanium fragments and then filtered through a 0.22μm filter into sterile vessels. The emulsion preparation (approximately 14 ml) was placed into a sterile syringe and then frozen at -70°C.

Example 4 Testing chyloremnant particles in humans

A volume of 12-15 ml of the emulsion mixture produced in Example 3 was injected intravenously into a group of normal-weight men and overweight men for assessment of remnant catabolism. Subjects were fasted overnight before the study. A total of 27 men were included in this study. Exclusion criteria included diabetes and hypercholesterolemia. Breath samples were collected at intervals over 24 hrs and enrichment with ¹³C was measured using isotope-ratio mass spectrometry (ABCA, Europa Scientific, Crewe, U. K.). The results are shown in Figure 2.

Results show that the enrichment of exhaled CO₂ with ¹³C peaked approximately 5 hrs after injection. Enrichment varied widely between individuals, and correlated significantly with BMI, waist circumference, insulin, leptin and fasting plasma HDL, LDL and TG concentrations. Compared with the control group (waist <95cm) the viscerally obese men (waist >95cm) showed significant differences in the breath test, as well as expected differences in BMI, and plasma insulin, leptin, cholesterol, HDL and TG concentrations. The results show that abnormal remnant metabolism forms part of the metabolic syndrome accompanying visceral obesity.

The present invention encompasses all modifications and adaptations apparent to those skilled in the art.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS

1. A non-radioactively labelled diagnostic composition for testing for the presence of or propensity for atherosclerosis and coronary artery disease and the like comprising components which mimic essential features of an exogenous lipoprotein transport particle, said composition being capable of metabolisation by normal physiological pathways such that at least one non-radioactively labelled metabolite is detectable in the blood or bodily waste of a patient being tested.

2. A non-radioactively labelled diagnostic composition as claimed in claim 1 wherein the exogenous lipoprotein transport particle mimics the chemical formulation of a chyloremnant.

3. A non-radioactively labelled diagnostic composition as claimed in claim 2 comprising a plurality of lipidic components selected in such a manner so as to mimic the essential features of a chyloremnant in which at least one of the components is a cholesteryl ester that is between about 10 to 40% of the total mass of lipid.

4. A non-radioactively labelled diagnostic composition as claimed in either claim 2 or 3 comprising a plurality of lipidic components selected in such a manner so as to mimic the essential features of a chyloremnant in which at least one of the components is a cholesteryl ester that is between about 10 to 30% of the total mass of lipid.

5. A non-radioactively labelled diagnostic composition as claimed in anyone of claims 2 to 4 comprising a plurality of lipidic components selected in such a manner so as to mimic the essential features of a chyloremnant in which at least one of the components is a cholesteryl ester that is between about 15 to 25% of the total mass of lipid.

6. A non-radioactively labelled diagnostic composition as claimed in any one of claims 2 to 5 comprising a plurality of lipidic components selected in such a manner so as to mimic the essential features of a chyloremnant in which at least one of the components is a cholesteryl ester that is between about 16.5 to 22.5% of the total mass of lipid.

7. A non-radioactively labelled diagnostic composition for testing the presence of or propensity for atherosclerosis and coronary artery disease and the like, said composition comprising:

wherein the composition is capable of metabolisation by normal physiological pathways such that at least one non-radioactively labelled metabolite is detectable in the blood or bodily waste of a patient being tested.

8. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 7 wherein the non-radio active label is ¹³C.

9. A non-radioactively labelled diagnostic composition as claimed in claim 8 wherein the amount of label within the diagnostic compositions is such that the isotope enrichment ratio of ¹³C relative to ¹²C in the CO₂ of the expired breath of a patient is at least measurable.

10. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 9 in which the composition comprises particles of a diameter of between about 10 to 1000 nanometers (nm).

11. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 10 in which the composition comprises particles of a diameter of between about 10 to 200nm.

12. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 11 in which the composition comprises particles of a diameter of between about 20 to 60nm.

13. A non-radioactively labelled diagnostic composition as claimed in claims any one of 1 to 12 in which the composition comprises particles of a diameter of between about 30 to 40 nm.

14. A non-radioactively labelled diagnostic composition for testing the presence of or propensity for atherosclerosis and coronary artery disease and the like, said composition comprising:

(ii) the particles have an average size of approximately 35 nm;

15. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 14 in which the composition comprises triglycerides, phospholipids, cholesterol, and cholesteryl esters.

16. A non-radioactively labelled diagnostic composition as claimed in claim 15 in which there is present in the composition triglycerides in an amount of 20 to 95% of total lipids.

17. A non-radioactively labelled diagnostic composition as claimed in either claim 15 or 16 in which the triglycerides suitable for use are triolein (TO), soybean oil, safflower oil, or other suitable substitutes such as olive oil or corn oil triglycerides.

18. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 17 in which there is present in the composition phospholipids such as egg yolk phosphatidylcholine, soybean phosphatidylcholine, dioleoyl phosphatidylcholine.

19. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 18 in which the phospholipid is present in the composition in an amount of about 5-50% of total lipids.

20. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 19 in which there is present in the composition cholesterol in an amount of about 5 to 20% of total lipids.

21. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 20 in which cholesterol is present in an amount of about 5 to 15% of total lipids.

22. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 21 in which cholesterol is present in an amount of about 7 to 10% of total lipids.

23. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 22 in which there is present in the composition 16 to 23% cholesteryl ester, 30-60% triglycerides, 8-30% phospholipids and about 7 to 10% cholesterol.

24. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 23 in which there is present in the composition approximately 46.5% triolein, 16.5% cholesteryl oleate, 9.5% cholesterol and 27.5% egg phosphatidylcholine.

25. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 24 in which the components of the composition, including the labelled component are emulsified in a solution in water of glycerol or glycerol and salts.

26. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 25 in which the composition includes a small quantity of an anti- oxidant to preserve the lipid against oxidation.

27. A non-radioactively labelled diagnostic composition as claimed in any one of claims 15 to 26 in which at least one of the components carries the label.

28. A non-radioactively labelled diagnostic composition as claimed in any one of claims 3 to 27 in which the label is attached to the cholesteryl ester.

29. A non-radioactively labelled diagnostic composition as claimed in claim 28 in which the label is attached to the fatty acid moiety of the cholesteryl ester.

30. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 29 in which the label is one that allows for the measurement of the amount of metabolite present by mass spectrometry.

31. A non-radioactively labelled diagnostic composition as claimed in any one of claims 1 to 30 in which the labelled metabolite may be measured in the captured expired air of the patient.

32. A non-radioactively labelled diagnostic composition as claimed in claim 1 substantially as herein described with reference to the examples.

33. A method of determining the presence of or propensity for atherosclerosis and coronary artery disease and the like in a patient requiring such determination comprising administering to said patient a labelled diagnostic composition which mimics essential features of an exogenous lipoprotein transport particle, and measuring the quantity of labelled metabolite in the bodily waste or blood of said patient so as to determine the quantity of labelled diagnostic composition successfully metabolised by said patient.

34. A method as claimed in claim 33 in which the composition mimicking the essential features of the exogenous lipoprotein particle is delivered to the patient by injection.

35. A method as claimed in either claim 33 or 34 in which the composition is one which mimics the chemical formulation of a chyloremnant.

36. A method as claimed in claim 35 in which the composition is primarily lipidic in nature.

37. A method as claimed in any one of claims 33 to 36 in which the composition comprises 20-95% by weight of total lipids of triglycerides, 5-50% by weight of total lipids of phospholipids, 2-20% by weight of total lipids of cholesterol, and 10-40% by weight of total lipids of cholesteryl esters.

38. A method as claimed in claim 37 in which at least a small proportion of the components are labelled.

39. A method as claimed in any one of claims 33 to 38 in which the composition comprises 16-23% cholesteryl ester, 30-60% triglycerides, 8-30% phospholipids and 7-10% cholesterol.

40. A method as claimed in claims 33 to 39 in which the measurement of the quantity of labelled metabolite present in the blood or bodily waste of the individual is measured from the patient's expired air.

41. A method as claimed in claim 33 as hereinbefore described with reference to the examples.