WO2011004009A1

WO2011004009A1 - Use of creatinine as dilution agent to determine the bodily composition of a mammal

Info

Publication number: WO2011004009A1
Application number: PCT/EP2010/059900
Authority: WO
Inventors: Vincent Biouge; Herve Lefebvre
Original assignee: Royal Canin Sas
Priority date: 2009-07-09
Filing date: 2010-07-09
Publication date: 2011-01-13
Also published as: FR2947908A1

Abstract

Procedure to determine the bodily composition of a mammal, including the use of creatinine as a dilution agent.

Description

USE OF CREATININE AS DILUTION AGENT TO DETERMINE THE BODILY COMPOSITION OF A MAMMAL

The present invention concerns a procedure to determine the bodily composition of a mammal by measuring the distribution volume of previously administered exogenous creatinine. This procedure makes it possible to quickly determine the water content of a human or animal. From this water content and thanks to well established equations, one can calculate the fatty mass and lean mass of the human or animal.

The measurement of bodily composition is an essential item in evaluating the general state of mammals. It particularly enables an overall assessment of nutritional state as well as an analysis of variations in bodily composition. The measurement of weight and size alone is quite insufficient to determine the nutritional state of a mammal.

In fact, it is essential to be able to precisely determine water content, in terms of fatty mass and lean mass, in both humans and animals. This is particularly true in cases of excess weight. For example, during weight reduction in humans or animals, it is preferable to target a loss of fatty mass and save muscular mass. The mere weight measurement is then found to be quite insufficient.

Furthermore, among certain animals it may be very difficult to determine with any certainty the existence or extent of excess weight or obesity. This is particularly true, for example, among dogs, in view of the existence of a large number of breeds with very heterogeneous formats. The normal weight therefore varies greatly from one breed to another. A precise measurement of water content, in fatty mass and in lean mass, then constitutes a valuable diagnostic tool in order to determine the nutritional state of the animal.

The body consists of elements of very different kinds: water, proteins, fats, bones etc. Numerous techniques have been described to determine the bodily composition of humans and animals. However these techniques are often too imprecise or too heavy by using tools reserved to research which are not suited to the clinician, who must be in a position to make a fast diagnosis of bodily composition. One of the methods commonly used is the isotopic dilution method with deuterium. This is used to determine the bodily composition of humans and animals. Burkholder and Thatcher (Burkholder WJ and CD Thatcher, 1998) have particularly validated the use of deuterium oxide to determine the bodily composition of dogs. The measurement of the deuterium space is effective in measuring the water content and thus determining the content of fat, proteins and mineral elements by calculation.

In a very different field, creatinine is the marker most often used in the direct or indirect estimation of the glomerular filtration (GFR) rate in domestic carnivore nephrology. This is likewise the marker classically used for humans.

Creatinine is a small molecule which results from the dephosphorylation of creatinine phosphate and the internal dehydration of creatinine. These two molecules are mainly contained in the muscles. The conversion to creatinine is spontaneous, non- enzymatic and irreversible. It affects approximately 2% of total bodily creatinine on a daily basis and is practically constant over the course of time. The excretion of creatinine is solely renal: it is totally filtered by the glomerula, secreted in a negligible quantity by the tubules and not reabsorbed. A test of plasma clarity of exogenous creatinine has been developed and validated among dogs (Watson et al., 2002.). The urinary and plasma clarity of creatinine makes it possible to measure the GFR, which is considered as the best indicator of renal function.

However, the use of creatinine to measure bodily composition in humans or animals had never been described nor even envisaged.

The applicant has now surprisingly found that after the injection of deuterium or exogenous creatinine in a mammal, the distribution volumes of deuterium and creatinine are very closely correlated. This correlation is observed for different nutritional states (normal or obese bodies for example). Thus, it is possible to replace the deuterium with creatinine in the dilution methods, making it possible to calculate the bodily composition of animals by determining the distribution volume of a dilution agent.

From the determination of the creatinine space, one thus easily deduces the water content of the body in order to then evaluate the lean mass and fatty mass according to classical equations. The use of creatinine presents numerous advantages in relation to the use of deuterium or other isotopes. Creatinine is easy to use, presents no risk of toxicity and may be used cheaply as a dilution agent. Moreover, the techniques of dosage of creatinine are already well known by the professional.

In veterinary clinics, for example, creatinine is already frequently used to determine the GFR, which is a good indicator of renal function. These veterinary clinics thus often have apparatus or hardware that allow the rapid dosage of creatinine in the blood or plasma. These tools may be implemented in order to quickly and easily determine the bodily composition of a mammal. Most preferably the invention described below is an in vitro procedure.

The invention relates to a procedure in vitro to determine the bodily composition of a mammal, including the following stages:

a) measurement of the basal plasma concentration in creatinine on a blood sample previously taken from the fasting mammal before the administration of a dose of creatinine,

b) measurement of the plasma concentration in creatinine on at least one blood sample previously taken from the mammal after the administration of a dose of creatinine,

c) determination of the distribution volume of the creatinine administered to the mammal,

d) calculation of the bodily composition of the mammal from the distribution volume of creatinine determined at the previous stage.

Advantageously, at stage d) the bodily water mass of the mammal is equal to the distribution volume of the creatinine previously administered.

In a first mode of realisation, at stage d), one calculates the lean mass of the mammal from the distribution volume of the creatinine previously administered.

In another mode of realisation, at stage d), one calculates the fatty mass of the mammal from the distribution volume of the creatinine previously administered.

Preferably, at stage b), one measures the plasma concentration in creatinine on a blood sample previously taken from the mammal after the administration of a dose of creatinine between 40mg/kg and 160mg/kg, preferably a dose of 40mg/kg. Preferably, at stage b), one measures the plasma concentration in creatinine on a blood sample previously taken from the mammal after the administration of a dose of creatinine, either orally, by intravenous injection or by subcutaneous injection.

Preferentially, at stage b), one measures the plasma concentration in creatinine on a blood sample previously taken from the mammal after a balancing time of at least 30 minutes and preferably at least 45 minutes after the administration of a dose of creatinine.

According to a preferred mode of realisation, at stage d) the distribution volume of creatinine in the mammal is calculated by dividing the dose administered by the plasma concentration of exogenous creatinine.

The procedures according to the present invention advantageously make it possible to determine the bodily composition, bodily water mass, lean mass and/or fatty mass in humans.

Preferably, the procedures according to the invention make it possible to determine the bodily composition, bodily water mass, lean mass and/or fatty mass among dogs.

Preferably, the procedures according to the invention make it possible to determine the bodily composition, bodily water mass, lean mass and/or fatty mass among cats.

The invention also relates to the use of creatinine for the manufacture of a dilution agent to determine the bodily composition, bodily water mass, lean mass and/or fatty mass of a mammal.

The invention also has as its object, a dilution agent to determine the bodily composition, bodily water mass, lean mass and/or fatty mass of a mammal that includes creatinine.

The invention also relates to a computer programme recorded on a support in order to commission the procedure according to the invention. Preferably, this computer programme recorded on a support includes at least the following:

instructions for calculating the distribution volume of creatinine administered to the mammal,

instructions for the calculation of the bodily composition, bodily water mass, lean mass and/or fatty mass of the mammal, from the plasma concentration in creatinine and the previously recorded dose of creatinine administered.

The object of the invention is therefore that of the methods of diagnosis of the bodily composition of a mammal.

In a first mode of realisation, the invention relates to methods to determine the water content of a mammal including the following stages:

measurement of basal plasma concentration in creatinine on a blood sample taken from the fasting mammal before the administration of creatinine, administration of a dose of exogenous creatinine,

- measurement of the plasma concentration in creatinine on a blood sample taken from the mammal after the administration of creatinine,

determination of the distribution volume of the creatinine administered to the mammal,

determination of the water content of the mammal from the distribution volume determined at the previous stage.

In a second mode of realisation, the invention relates to methods to determine the lean mass content of a mammal including the following stages:

measurement of the basal plasma concentration in creatinine on a blood sample taken from the fasting mammal before the administration of creatinine, - administration of a dose of exogenous creatinine,

measurement of the plasma concentration in creatinine on a blood sample taken from the mammal after the administration of creatinine,

- determination of the lean mass content of the mammal from the distribution volume determined at the previous stage.

In a third mode of realisation, the invention relates to methods to determine the fatty mass content of a mammal including the following stages:

measurement of the basal plasma concentration in creatinine on a blood sample taken from the fasting mammal before the administration of creatinine, administration of a dose of exogenous creatinine, measurement of the plasma concentration in creatinine on a blood sample taken from the mammal after the administration of creatinine,

- determination of the fatty mass content of the mammal from the distribution volume determined at the previous stage.

Preferably, creatinine is administered orally, by intravenous injection or by subcutaneous injection.

Preferably, one measures the plasma concentration in creatinine on at least one blood sample taken from the mammal after the administration of creatinine. The plasma concentration in creatinine may thus be measured on blood samples, taken at regular intervals after the administration of creatinine.

Preferentially, one measures the plasma concentration in creatinine on a blood sample taken from the mammal at least 45 minutes after the administration of creatinine.

The invention thus concerns a procedure to determine the bodily composition of a mammal by using creatinine as a dilution agent. The procedure is based on the determination of the distribution volume of exogenous creatinine previously administered to the mammal.

"Determination of bodily composition" signifies the determination of at least the water content, lean mass content and/or fatty mass content.

The distribution may be defined as the process of distribution of creatinine in all the tissues and organs following its administration to the mammal. The distribution volume Vd is defined as the apparent volume in which the creatinine is distributed in order to be in equilibrium with the plasma concentration C. Preferably, the

distribution volume of exogenous creatinine is calculated from the plasma

concentration of exogenous creatinine once this equilibrium is achieved.

The distribution volume of creatinine, also known as creatinine 'space' may be measured and calculated according to classical pharmacokinetic methods.

In the present invention, it has been surprisingly found that the distribution volume of creatinine makes it possible to directly deduce the water content of the body. From the water content, the lean mass and fatty mass are likewise deduced according to classical equations abundantly described in literature.

Given that creatinine is naturally present in the blood of mammals, it is necessary to previously determine the creatininemia, that is, the basal creatinine concentration in the blood. This basal plasma concentration shall then be taken from the plasma concentrations in creatinine measured after the administration of exogenous creatinine. It is thus possible to measure the plasma concentration of exogenous creatinine. These techniques are well known by the professional.

Furthermore, certain exterior factors may affect the value of the plasma concentration of creatinine. Food intake is the major factor of variation, in fact, creatininemia increases by an average of 45% in the first few hours following a meal consisting of cooked meat and then remains high for 12 hours. This increase is due to the composition of the meal and there are individual differences in the variation of creatininemia after eating. The professional will therefore understand that in order to obtain a reliable measurement of creatinine space, measurements of plasma concentrations must be carried out on blood samples previously taken from a mammal having received a dose of creatinine while fasting.

In order to determine the distribution volume, exogenous creatinine is previously administered to the mammal by intravenous injection (IV), subcutaneous injection, orally or by any other appropriate method.

The dose of creatinine administered stands between 40 mg/kg and 160 mg/kg. Preferably, the dose of creatinine previously administered is around or is 40mg/kg. Upon administration of a dose in the order of 40mg/kg, it is not necessary to further dilute the plasmas in order to measure their concentration in creatinine.

After administering the creatinine, it is distributed in the body. Before measuring the plasma concentration in exogenous creatinine, it is therefore preferable to allow sufficient time to elapse for the distribution of the creatinine that is, sufficient time to balance the plasma concentration of creatinine. This balancing time is between 45 minutes and 240 minutes. Preferably, this balancing time is at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours and at least 4 hours.

The plasma concentration of creatinine may be determined by a series of blood samples taken over the course of time following the administration of creatinine to the mammal. The establishment of a complete kinetic of the creatinine plasma concentration makes it possible to measure the distribution volume of creatinine with great precision. However, for numerous diagnostic applications, it may be sufficient to measure the plasma concentration in a single blood sample taken at a sufficiently long time after the administration of creatinine.

In the examples, it is thus shown that the distribution volume of creatinine may be determined sufficiently reliably from the measurement of plasma concentration on a single blood sample taken after the administration of creatinine. Preferably, the sample should be taken at least 45 minutes after the administration of creatinine.

The plasma concentration of creatinine is measured according to known techniques described in literature. One shall quote in particular the methods of colour- coded doses as well as methods of enzymatic dosage. For example, the Jaffe method can be used, which is based on the use of the ion picrate or, preferentially, an enzymatic method using amidohydrolase creatinine or creatininase. However, any method that allows the dosage of creatinine in the plasma may be commissioned in the procedures of the present invention.

The plasma concentration in creatinine measured on a blood sample makes it possible to determine the distribution volume of the creatinine administered. This distribution volume is obtained by dividing the administered dose by the plasma concentration. The distribution volume of the creatinine is determined by the formula Vd=D/C in which D represents the dose of exogenous creatinine previously administered to the mammal, and C represents the plasma concentration of creatinine from which the basal plasma concentration in creatinine has been removed.

By using deuterium as a dilution agent, one determines the distribution volume of deuterium, then one uses classical equations to deduce the water content, lean mass content and fatty mass content. The present invention is based on the unexpected observation according to which the distribution volume of creatinine is correlated in linear fashion with the distribution volume of deuterium. Thus, the determination of the distribution volume of creatinine makes it possible to deduce the water content, and then the lean mass content and fatty mass content, by using the equations already used for deuterium which are well known by the professional. These classical equations make it possible to precisely estimate the bodily composition from the distribution volume of a dilution agent such as deuterium. The reliability of the measurement of bodily composition from the distribution volume of a dilution agent has been particularly validated among dogs (Burkholder WJ and CD Thatcher, 1998), in humans (Forbes GB, 1994) and among cats (Backus RC et al,

2000).

In a first mode of realisation, one only measures the concentration of the dilution agent (creatinine) on a sample of plasma previously taken. Preferably, the plasma example is previously taken once the creatinine concentration in the blood has reached equilibrium. The bodily water volume is then calculated from the distribution volume according to the following equation:

Vwater=Vd=D/C

in which Vd represents the distribution volume of creatinine which is equal to the bodily water volume (or the bodily water mass), D is the dose of creatinine administered and C is the plasma concentration measured (from which one must deduct the basal concentration in creatinine). This distribution volume may be expressed in ml or grams, as 1 ml of water = 1 g.

In a second mode of realisation, one carries out multiple measurements of concentration of the dilution agent (creatinine) on different samples of plasma previously taken. The bodily water volume is then calculated from the distribution volume according to the following equation:

Vwater=Vd= D x AUMC/AUC²

in which Vd represents the distribution volume of creatinine which is equal to the bodily water volume (or the bodily mass of water), D is the dose of creatinine administered, AUC (area under the curve) is the surface under the curve which constitutes the concentration according to time (measured duration of 0 to 240 minutes, for example) and AUMC (first curved moment) which is the surface under the curve of concentration from time 0 to infinity.

These methods of calculation and classical equations of pharmacokinetics are described, for example, by Gibaldi and Perrier (1982).

The lean mass is directly calculated from the bodily water mass by using the following equation: Lean mass (g)= mass in water (g)/ constant

The constants used are well known by the professional and broadly described in literature. Typically, the constant in humans = 0.73 (Forbes GB, 1994) and the constant among dogs and cats = 0.744 (Ferrier L et al, 2002, Backus RB et al, 2000).

The fatty mass is directly deduced from the lean mass according to the following equation:

Fatty mass (g) = weight (g) - lean mass (g) in which the weight designates the live weight of the man or mammalian animal.

The procedures of the present invention may be applied to the determination of the bodily composition of any mammal.

In a first mode of realisation, the procedures of the present invention make it possible to determine the bodily composition in humans.

In a second mode of realisation, the procedures of the present invention make it possible to determine the bodily composition of mammalian animals such as, in particular, dogs or cats.

The present invention refers to Figures 1 to 11, as follows:

Figure 1 is a comparison of the distribution volume of creatinine with the distribution volume of deuterium;

Figure 2 is a comparison of the mean distribution volume of creatinine with the mean distribution volume of deuterium from the results in Table 2;

Figures 3 to 10 illustrate the kinetics of plasma concentration in creatinine before and after weight gain;

Figure 11 is a comparison of distribution volume using a method with 2 sample-takings versus the method with complete kinetics.

EXAMPLES

Example 1 : Comparative study of the distribution volume of deuterium and creatinine among normal and then obese dogs: Strategy with several sample takings.

1. Determination of bodily water volume by multiple measurement of water intake A group of 6 beagle dogs in optimal physical condition was used; in the morning of the study, the dogs having received their last meal the morning before, 2 mL of blood was taken from the jugular vein and the plasma was collected for the basal measurement of creatinine and D₂O. After placing a catheter in the cephalic vein, the doses OfD₂O (500 mg/kg of live weight, 99.9% D₂O containing 0.9 %NaCl), and Creatinine (40 mg/kg mixed weight at 5 mL of physiological serum) is injected and the catheter flushed with physiological serum. Blood samples were then taken at 2, 5, 10, 15, 30, 45, 60, 90, 120, 240, 360 and 480 minutes after the injection. The weight of the animal on the day of the trial and the exact times when the blood samples were taken, are recorded. The plasma is collected after centrifuging the blood samples, and it is kept at 4°C awaiting the measurements of concentration of D₂O and creatinine. The dogs are then fed according to their own appetite with a commercial dog food rich in fatty matter so as to stimulate weight gain. Once the animals display an obese physical condition, the same protocol is repeated.

Table 1 illustrates the variations of creatinine concentration in the plasma of dog A in optimal physical condition and after weight gain.

Figures 3 to 10 illustrate the kinetics of plasma concentrations in creatinine before and after weight gain. Table 1 and curves 3-10 illustrate the level of basal creatinine at time 0, the rapid increase of creatinine plasma following the injection of exogenous creatinine, the progressive fall of the concentration in plasma creatinine until 480 minutes after the injection.

Similar kinetics curves have been observed for the evolution of the concentration of D₂O.

2. Calculation of bodily water content

From the kinetic curves of the concentrations of deuterium and creatinine in plasma, it is possible to calculate the distribution volume of these markers in the body. It has been confirmed in humans, dogs, cats and other mammals, that the distribution volume of D₂O is equivalent to the volume of bodily water.

The method of calculation of the distribution volume is a method currently used in pharmacokinetics; it is a non-compartmental method based on the statistical theory of moments (Gibaldi and Perrier, 1982). Briefly, this method determines the distribution volume in accordance with the following equation:

Distribution volume = Dose administered x AUMC /AUC²

Table 2 summarises the distribution volumes of deuterium and creatinine before and after weight gain. Results

Vd Vd

deuterium creatinine Mean Delta

Dog Status mL/kg mL/kg mL/kg mL/kg

A Normal 594.2 553.2 573.7 -41

C Normal 547.4 501.8 524.6 -45.6

E Normal 560.4 536.2 548.3 -24.2

F Normal 566.9 545.8 556.35 -21.1

G Normal 558.8 523.9 541.35 -34.9

H Normal 557.2 602.3 579.75 45.1

A Obese 423.3 419.5 421.4 -3.8

C Obese 394.6 389.8 392.2 -4.8

E Obese 408.1 407.9 408 -0.2 F Obese 422 428 425 6

G Obese 399.7 398.1 398.9 -1.6

H Obese 439.8 403.4 421.6 -36.4

Mean 489.4 475.8 482.6 -13.5

AND 79.7 75.2 25.7

These data show that the distribution volume of exogenous creatinine correlates very well with the distribution volume of creatinine in figure 1. They also show that the mean distribution volume of creatinine is approximately 97.2 % of the distribution volume of D₂O (Table 2 and Figure 2). As the D₂O over-estimates the volume of bodily water by 2-3% among dogs (see above), the distribution volume of creatinine satisfactorily represents the volume of bodily water.

3. Calculation of bodily composition in lean mass and fatty mass

Once the volume (or weight of bodily water) has been determined by the above method, it is easy to calculate the composition in lean mass by methods well known by the professional. (Forbes GB, 1994, Ferrier L et al, 2002, Backus RB et al 2000).

In the above example (Table 2), the mean distribution volume of deuterium is 489.4 ml/kg of weight; to determine the volume of bodily water one must use a correction factor of 0.98 among dogs (see above), hence the volume of bodily water = 489.4*0.98= 479.22 mL of water/kg live weight. We unexpectedly found that the mean volume of the distribution of creatinine gave a very similar value, that is, 475.8 mL of water/kg live weight. To determine the proportion of lean mass, one uses the following equation:

Proportion of lean mass = Mass of bodily water in g/kg/0.744 thus in our example: 475.8/0.744 = 639.5 g/kg of weight. The proportion of fatty mass may be calculated differently: Proportion of fatty mass = 1000 - proportion of lean mass = 1000 - 639.5 = 360.5 g/kg of live weight.

Example 2: Comparative study of the distribution volume of deuterium and creatinine among normal then obese dogs: Strategy limited to 2 samples taken in order to obtain an estimate of the distribution volume of creatinine after intravenous administration of exogenous creatinine.

The principle of the study is the same as for the strategy of multiple sample-taking, but this time only 2 samples are taken: one before the administration of creatinine to determine the basal value (TO) and a second one 45 minutes after administration. As in the first example, the dose injected is 40 mg (354 μmole)/kg of weight. To determine the distribution volume of creatinine (Table 3), one divides the injected dose (μmole or mg) by the concentration (C) of creatinine in the plasma (μmole/L or mg/L) observed at 45 minutes, from which the basal value of pre-administration

(base) has been removed. If the concentration is expressed in mg/L, the dose must be expressed in mg.

Table 3 - Estimation of distribution volume at 45 minutes.

When one compares the distribution volume with the method of 2 sample-takings versus the method with complete kinetics (table 4 and fig.11), one observes a significant correlation.

Table 4 - Comparison of distribution volume estimated from the plasma concentration of creatinine measured at 45 minutes (Vd45) and the distribution volume (Vss) obtained with complete kinetics.

The correlation between Vss and Vd 45 is shown in figure 11.

The following equation thus makes it possible to estimate the Vss from Vd 45 :

Vss estimated = (Vd45-287.93)/0.7545 (equation 1) (R2=0.4481)

or again Vss estimated = 1.325 * Vd 45 - 380.782

One may note (Table 5) that this equation makes it possible to estimate the real Vss, and thus the bodily water content, among dogs in optimal physical condition with a discrepancy of -17.6 to 9.3% and among obese dogs with a discrepancy of -28.5 to

+37.1%. These variations are acceptable within the framework of clinical practice.

Table 5 - Estimation of the Vss from Dose/C-base

Vss= Vss calculated with complete kinetics; Dose/C-base: distribution volume estimated from the concentration at 45 minutes; V estimated: Vss estimated from equation 1. Dose/C-

Vss base

Status Dog (mL/kg) (mL/kg) V estimated Delta (%)

Normal A 553.2 746 607 9.8

Normal C 501.8 625 447 -10.9

Normal E 536.2 621 442 -17.6

Normal F 545.8 711 560 2.7

Normal G 523.9 678 517 -1.4

Normal H 602.3 785 658 9.3

Obese A 419.5 584 393 -6.3

Obese C 389.8 691 535 37.1

Obese E 407.9 531 322 -21.2

Obese F 428 599 413 -3.5

Obese G 398.1 503 285 -28.5

Obese H 403.4 689 531 31.7

Mean 476 647 476

SD 75 85 112

Among healthy dogs, the discrepancy between Vestimated and Vss varies from -17.6 to 9.3%

Among obese dogs, the discrepancy varies from -28.5 to +37.1%

From the estimation of the Vss, the water content in lean and fatty mass is calculated in the same way as for example 1.

REFERENCES

Backus RC, Havel PJ, Gingericj RL, Rogers QR. Relationship between serum leptin immune-reactivity and body fat mass as estimated by use of a New gas-phase Fourier- Transform infrared spectroscopy deuterium dilution method in cats. AJVR 2000, 61 :796-801. Burkholder WJ and Thatcher CD, AJVR, Vol. 59, No. 8, 1998

Ferrier L, Robert P, Dumon H, Martin L and Nguyen P. Evaluation of body composition of dogs by isotopic dilution using a low cost technique, Fourier- Transform Infrared spectroscopy. J Nutr 2002, 132: 1725 S-1727S.

Forbes GB, Body composition: influence of nutrition, disease, growth and ageing. In Modern Nutrition in Health and Disease, Eds Shils ME, Olson JA, Shike M, Philadelphia: Lea & Febiger, 2004; 781- 801.

Gibaldi M and Perrier D, Noncompartemental analysis based on statistical moment theory. In Pharmacokinetics, Marcel Dekker, Inc. New York, 1982, 409-417.

Watson et al, J. Vet Intern Med, 16:22-33, 2002

Claims

1. Procedure in vitro to determine the bodily composition of a mammal including the following stages:

a) measurement of basal plasma concentration in creatinine on a blood sample previously taken from the fasting mammal before the administration of a dose of creatinine,

b) measurement of plasma concentration in creatinine on at least one blood sample previously taken from the mammal after the administration of a dose of creatinine,

2. Procedure in vitro according to claim 1 in which at the stage d) the bodily water mass of the mammal is equal to the distribution volume of the creatinine previously administered.

3. Procedure in vitro according to claim 1 in which, at stage d), one calculates the lean mass of the mammal from the distribution volume of the creatinine previously administered.

4. Procedure in vitro according to claim 1 in which, at stage d), one calculates the fatty mass of the mammal from the distribution volume of the creatinine previously administered.

5. Procedure in vitro according to one of the previous claims in which, at stage b), one measures the plasma concentration in creatinine on a blood sample previously taken from the mammal after the administration of a dose of creatinine between 40mg/kg and 160mg/kg, preferably a dose of 40mg/kg.

6. Procedure in vitro according to one of the previous claims in which, at stage b), one measures the plasma concentration in creatinine on a blood sample previously taken from the mammal after the administration of a dose of creatinine, either orally, by intravenous injection or by subcutaneous injection.

7. Procedure in vitro according to one of the previous claims in which, at stage b), one measures the plasma concentration in creatinine on a blood sample previously taken from the mammal after a balancing time of at least 30 minutes and preferably at least 45 minutes after the administration of a dose of creatinine.

8. Procedure in vitro according to claim 7 in which, at stage d) the distribution volume of the creatinine in the mammal is calculated by dividing the dose administered by the plasma concentration in exogenous creatinine.

9. Procedure in vitro according to one of the previous claims to determine the bodily composition, bodily water mass, lean mass and/or fatty mass in humans.

10. Procedure in vitro according to one of the claims 1-8 to determine the bodily composition, bodily water mass, lean mass and/or fatty mass in dogs.

11. Procedure in vitro according to one of the claims 1-8 to determine the bodily composition, bodily water mass, lean mass and/or fatty mass in cats.

12. Use of creatinine to manufacture a dilution agent to determine the bodily composition, bodily water mass, lean mass and/or fatty mass of a mammal.

13. Dilution agent to determine the bodily composition, bodily water mass, lean mass and/or fatty mass of a mammal featuring the inclusion of creatinine.

14. Computer programme recorded on a support for the commissioning of the procedure according to one of the claims 1-11.

15. Computer programme recorded on a support according to claim 14 including at least:

instructions for the calculation of the distribution volume of the creatinine administered to the mammal,

instructions for the calculation of bodily composition, bodily water mass, lean mass and/or fatty mass of the mammal, from the previously recorded plasma concentration in creatinine and the dose of creatinine administered.