DE102018214660A1 - Diagnostic method for diseases that are associated with a reduced bone density - Google Patents

Abstract

The invention relates to a method for diagnosing a disease which is associated with a reduced bone density and / or calcium loss, comprising the following steps: a) determining isotope ratios or quantitative ratios of alkaline earth metal fractions in a sample of urine or blood or stool, b) thereby Determining at least one sample value, and c) comparing the at least one sample value with a threshold value, the threshold value being selected independently of one or more factors caused by the individual patient, and a test kit for use in carrying out the method.

Description

The invention relates to the diagnosis and / or the exclusion of diseases which are associated with a reduced bone density, such as, for example, osteoporosis, osteomalacia, multiple myeloma and / or renal dysfunction.

Diseases associated with reduced bone density, such as osteoporosis, osteomalacia, multiple myeloma and / or renal dysfunction, represent a major problem worldwide. According to the WHO, osteoporosis alone is one of the 10 most common diseases.

The osteoporosis occurs increasingly from a certain age and about four times more often in women than in men.

In Germany, the main diagnoses of osteoporosis with pathological fracture even increased by more than 20% between 2010 and 2016.

It therefore seems very sensible to carry out more diagnoses (serial examinations), especially in the risk group concerned, in order to recognize the risk of developing osteoporosis early and to take countermeasures. This could also minimize the risk of undetected osteoporosis and the possibly resulting health damage (broken bones, postural damage).

Even in view of the high number of diseases, a screening procedure that could be used within the framework of preventive medicine and so can not separate those affected from the potentially affected patients at a very early stage would be very desirable.

It would also be very sensible to carry out diagnoses accompanying therapy, in order to identify a therapy for diseases that do not work as desired, which is associated with a reduced bone density, and ideally also to learn more about the causes of the disease and to be able to take more suitable measures.

The standard method currently used to diagnose and control osteoporosis and other bone metabolism disorders with an increased risk of bone fracture is the X-ray-based DXA or DEXA (Dual-energy X-ray absorptiometry). Disadvantages here are the resulting radiation exposure for the patient and the poor comparability of the determined values. Therefore, no absolute values can be given in the findings, but only a deviation from the age and gender-specific average is determined and compared with the standard deviation.

The US 2013/0115650 A1 suggests using the ratio of ⁴⁴ Ca / ⁴² Ca as a biomarker to diagnose diseases associated with changes in the mineral balance of the human skeleton. For a statement, however, a baseline must first be determined for each individual (first sample) shortly after the start of therapy, another sample is taken and measured.

Skulan, Joseph; et al; "Natural Calcium Isotopic Composition of Urine as a Marker of Bone Mineral Balance" Clinical Chemistry, 53, 1155-1158 (2007) propose to measure the calcium-isotope ratio δ ^44/40 Ca in the urine at different times over a longer period of time in order to monitor the mineral ^{balance of} the skeleton and to detect possible "bone loss" due to a longer bed rest.

The US 2014/0273248 A1 involves checking the mineral balance of the human skeleton in cancer patients. Here, too, a baseline must first be determined, then further samples are taken after the start of therapy consisting of the administration of aromatase inhibitors.

Diagnostic methods that require normalization to the individual patient through multiple measurements (e.g. by recording a baseline) are rather unsuitable for use in preventive medicine, especially in the context of a screening procedure.

It is therefore an object of the invention to provide a diagnostic method for diseases associated with a reduced bone density, in which one is stressful for the patient X-rays and extensive standardization on the individual patient, for example using a baseline, can be dispensed with.

1. a) Bestimmen von Isotopenverhältnissen oder Mengenverhältnissen von Erdalkalimetall-Anteilen in einer Probe von Urin oder Blut oder Stuhl,
2. b) hierdurch Ermitteln mindestens eines Probenwertes, und
3. c) Vergleich des mindestens einen Probenwertes mit einem Schwellenwert,

wobei der Schwellenwert unabhängig gewählt ist von einem oder mehreren durch den individuellen Patienten bedingten Faktoren.The object of the invention is achieved by a method for diagnosing a disease which is associated with a reduced bone density and / or calcium loss, comprising the following steps:

1. a) determining isotope ratios or quantitative ratios of alkaline earth metal fractions in a sample of urine or blood or stool,
2. b) thereby determining at least one sample value, and
3. c) comparison of the at least one sample value with a threshold value,

the threshold value being chosen independently of one or more factors determined by the individual patient.

The sample values are determined in previously taken blood and / or urine and / or stool samples.

In the context of the present invention, the term that the threshold value is selected independently of one or more factors caused by the individual patient means, in particular, that neither a patient-related baseline with respect to the sample values to be determined has to be determined, nor other factors such as, for example, a nutritional plan to be followed before the actual diagnosis or similar factors or measurements of previous values of the individual patient are carried out and are also not included in the diagnostic process. In other words, the threshold value, depending on the type of urine, blood or stool sample, is a fixed numerical value.

Advantageously, a method for diagnosing diseases associated with reduced bone density and / or calcium loss can now be made available for the first time, which is both significantly easier to use and can therefore be used in preventive care. The elimination of previous determinations of baselines and other influencing factors, such as nutritional control and nutritional protocols, which the individual patient has so far been required to provide even before the actual diagnosis has been carried out, considerably facilitates the implementation and use of the method permits widespread use and can be used in preventive diagnosis, especially for older women, even without suspicion. A significant cost reduction for the simplified procedure is also an important factor in the further application and distribution, so that for the first time such "screening" is available for a much larger group of patients.

The sample values are determined isotope ratios or quantitative ratios of alkaline earth elements. In particular, the values are isotope ratios of calcium (Ca) or the quantitative ratio of the alkaline earth elements of calcium (Ca) to strontium (Sr).

The sample values are preferably isotope ratios or mass ratios of alkaline earth elements determined by mass spectrometry.

In a preferred embodiment of the method, the isotope ratios of calcium (Ca) are determined in step a).

In a further preferred embodiment of the method, the quantitative ratio of the alkaline earth elements of calcium (Ca) to strontium (Sr) is determined in step a).

In one aspect of the invention, a fixed threshold value, which is independent of the individual and which was previously determined depending on the type of sample, serves as a comparison value in step c).

In one aspect of the invention, the threshold value in step c) for the isotope ratio ⁴⁴ Ca / ⁴² Ca in a blood sample after a δ notation is δ ^44/42 Ca _blood = -0.85 ± 0.06% o.

In another aspect of the invention, the threshold value in step c) for the isotope ratio ⁴⁴ Ca / ⁴² Ca in a urine sample after a δ notation is δ ^44/42 Ca _urine = 0.23 ± 0.06 ‰.

mit m3 > m2 > m1, wobei m3, m2, m1 die Massenzahlen repräsentieren, auf andere Isotopenverhältnisse des Calciums übertragen werden.The threshold can be according to the formula: $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

with m3>m2> m1, where m3, m2, m1 represent the mass numbers, are transferred to other isotope ratios of calcium.

In a further aspect of the invention, the threshold value in step c) for the quantitative ratio of the alkaline earth elements of calcium (Ca) to strontium (Sr) is 1772 ± 250 molca / molsr.

In a preferred embodiment of the invention, a sample value is determined as δ ^44/42 Ca _urine and a further sample value as δ ^44/42 Ca _blood in step b) and in step c) the difference between the two sample values is compared with a threshold value by an additional one To make a statement about kidney function.

In a further preferred embodiment of the invention, a sample value as δ ^44/42 Ca _stool and a further sample value as δ ^44/42 Ca _{blood are} determined in step b) and in step c) the difference between the two sample values is compared with a threshold value, in addition to make a statement about bowel function.

In one aspect of the invention, the disease associated with decreased bone density and / or calcium loss is osteoporosis, osteomalacia, multiple myeloma and / or renal dysfunction.

In a preferred embodiment of the method, the quantitative ratios in step a) are determined as molar ratios of the alkaline earth metal fractions in the sample or as mass ratios of the alkaline earth metal fractions in the sample.

A further preferred embodiment of the method is characterized in that if a sample value is determined as δ ^44/42 Ca _urine in step b) and the comparison with the threshold value after a δ notation of δ ^44/42 Ca _urine in step c) 0.23 ± 0.06 ‰ shows that the sample value is larger, the presence of an osteoporosis disease can be excluded as unlikely.

A further preferred embodiment of the method is characterized in that if a sample value is determined as δ ^44/42 Ca _blood in step b) and the comparison with the threshold value after a δ notation of δ ^44/42 Ca _blood in step c) -0.85 ± 0.06% o shows that the sample value is larger, the presence of an osteoporosis disease can be excluded as unlikely.

One aspect of the invention is a test which, by measuring the isotope ratio of calcium isotopes, can determine whether postmenopausal women have no diseases associated with reduced bone density, such as e.g. Osteoporosis, osteomalacia and / or multiple myeloma or whether there is an increased probability of a positive diagnosis of one of these diseases.

In a further aspect of the invention, the isotope ratio ⁴⁴ Ca / ⁴² Ca is determined in a previously taken blood sample and standardized to an international standard according to a δ notation.

The δ ^{m3 / m2} Ca values can also be transferred to other Ca isotope ratios using the following formula (for example with the participation of ⁴⁰ Ca, ⁴¹ Ca, ⁴⁶ Ca, ⁴⁸ Ca, ⁴³ Ca): $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

With m3> m2> m1

According to the invention, the value determined in this way is compared to a threshold value (for δ ^44/42 Ca _blood : -0.85 ± 0.06% o), if the values are above this threshold value there are no diseases associated with a reduced bone density, such as, for example, osteoporosis, osteomalacia and / or multiple myeloma, if the values are below the threshold, there is an increased risk of a positive diagnosis of a disease that is associated with a reduced bone density.

It is therefore possible to arrive at a diagnosis result on the basis of a single sample. The threshold value of δ ^44/42 Ca _blood = -0.85 ± 0.06% o was determined on the basis of a registered study (NCT02967978) following the Helsinki criteria "for good clinical practice" in the 100 female postmenopausal test subjects who were between 50 and 75 years old, determined (Osteogeo study). Due to the variation of the measured values, the value is to be assigned a statistical uncertainty of ± 0.06% o, ie from -0.79% o to -0.91 ‰ there is a measurement-related uncertainty as to whether the patient is suffering from bone loss or not. The latter must be taken into account in the diagnosis or diagnosis (see . and ).

The threshold value for δ ^44/42 Ca _blood can also be transferred to other Ca isotope ratios using the following formula (for example with the participation of ⁴⁰ Ca, ⁴¹ Ca, ⁴⁶ Ca, ⁴⁸ Ca, ⁴³ Ca): $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

With m3> m2> m1

The result is very meaningful, the sensitivity of the procedure is 94.4%, the negative likelihood factor is 0.1, which means that women whose measured values are above the threshold value are only 0.1 times more likely to have a positive osteoporosis finding than a healthy person to get.

Before the study, it could not be expected that a diagnosis could only be possible on the basis of a value and with this informative value, rather it had to be assumed that the calcium isotope content of the food consumed would also have an influence on the result. This would mean that standardization to the individual person by measuring different samples of a person and / or possibly several measurements if a diet which controls the calcium isotope content of the food would be necessary.

The shows the measured calcium isotope of the food, stool, blood and urine of the 100 postmenopausal women from the Osteogeo study. Although the nutritional behavior of the 100 women varies greatly, their calcium isotope content is very constant and can be estimated with δ ^44/42 Ca: -0.43 ± 0.05% o within narrow limits. This also means that a detailed query of the food consumed is not absolutely necessary for a diagnosis, since a generally known value can be assumed, even if there is a special nutritional situation such as vegetarian or vegan.

Furthermore, the Osteogeo study showed that the influence of calcium from food on the calcium isotope composition of the blood is small, since the absorption rate of calcium from the intestine is a maximum of 30% up to approx. 10%. This means that only about 0.3 to 0.15 g of the daily calcium in the order of about 1 gram of calcium per day (gca / day) is absorbed. However, this corresponds to only approx. 2.2% to 1.1% of the daily amount of calcium (13.18 g Ca / day) from the kidneys (approx. 6.67 g Ca / day, approx. 50%) and from the bones (6.36 g Ca / day, approx. 48%). It follows from this that any isotopic variations in calcium in food are small to negligible due to the mass balance.

Although the calcium isotope of food was almost constant for all subjects in the study, the calcium isotope values for blood, urine and stool are different between the different organs due to the body's own fractionation processes.

The calcium flows between the various organs in healthy postmenopausal women determined in the course of the Osteogeo study can be graphically represented as follows:

The following graphic shows the calcium flows between the organs in postmenopausal women who suffer from osteoporosis. The differences in calcium flows between the compartments involved are marked by a broken line.

The invention has now recognized that this profound knowledge about the calcium flows between the organs in healthy and osteoporotic women can be implemented in a test method in which the absence of a disease associated with a reduced bone density, such as e.g. Osteoporosis, osteomalacia and / or multiple myeloma (negative test) and at the same time increased probability of receiving a positive diagnosis of the diseases mentioned. It is thus possible to arrive at a diagnosis result on the basis of a single blood sample.

Another aspect of the invention is a test in which, by measuring the isotope ratio of calcium isotopes in a previously taken urine sample, it can be determined whether postmenopausal women have no diseases associated with reduced bone density, e.g. Osteoporosis, osteomalacia and / or multiple myeloma or whether there is an increased probability of a positive diagnosis of one of these diseases.

The isotope ratio ⁴⁴ Ca / ⁴² Ca is determined in a previously taken urine sample and standardized to an international standard according to a δ notation.

According to the invention, the value determined in this way is compared to a threshold value (for δ ^44/42 Ca _urine 0.23 ± 0.06% o), if the values are above this threshold value there is no osteoporosis, if the values are below the threshold value, there is an increased risk of positive osteoporosis diagnosis.

It is therefore possible to arrive at a diagnosis result on the basis of a single sample. The threshold value of δ ^44/42 Ca _urine 0.23 ± 0.06% o was determined on the basis of a study in 100 female test subjects who were between 50 and 75 years old.

The threshold value for δ ^44/42 Ca _urine can also be transferred to other Ca isotope ratios using the following formula (for example with the participation of ⁴⁰ Ca, ⁴¹ Ca, ⁴⁶ Ca, ⁴⁸ Ca, ⁴³ Ca): $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

With m3>m2> m1

The sensitivity of the procedure is 72.2%, the negative likelihood factor is 0.41, which means that women whose measured values are above the threshold value are only 0.41 times more likely to get a positive osteoporosis finding than a healthy person.

Before the study, it could not be expected that a diagnosis could only be possible on the basis of a value, rather it had to be assumed that the calcium isotope content of the food consumed would also have an influence on the result. This would mean that standardization to the individual person by measuring different samples of a person and / or possibly several measurements if a diet which controls the calcium isotope content of the food would be necessary.

The invention has now recognized that this profound knowledge about the calcium flows between the organs in healthy and osteoporotic women can be implemented in a test method in which the absence of a disease associated with a reduced bone density, such as e.g. Osteoporosis, osteomalacia and / or multiple myeloma (negative test) and at the same time increased probability of receiving a positive diagnosis of the diseases mentioned. It is therefore possible to arrive at a diagnosis result on the basis of a single urine sample.

The possibility of diagnosis on the basis of a single sample from an individual is a very surprising result. The existence of the threshold value of δ ^44/42 Ca _urine 0.23 ± 0.06 ‰ and δ ^44/42 Ca _blood -0.85 ± 0.06% o could not be expected.

Another aspect of the invention is a test in which, by measuring the ratio of the alkaline earth elements calcium (Ca) to strontium (Sr) in urine (Ca / Sr) _urine, the ability to resorb Ca and thus the likelihood of the presence of one Diseases associated with reduced bone density, such as osteoporosis, osteomalacia and / or multiple myeloma, are characterized. The result is particularly surprising against the background, since the ratio of Ca and magnesium (Mg), also an alkaline earth element, does not have this correlation.

A statistically significant correlation between the (Ca / Sr) _urine values and the DXA values could be determined from the ROC analysis (Receiver Operating Characteristics). The DXA method is the currently valid standard method for determining bone density and diagnosing osteoporosis.

A (Ca / Sr) _urine threshold value was determined, which allows patients with osteoporosis to be distinguished from healthy patients. The threshold was 1772 ± 250 molca / molsr, which separates sick from healthy patients.

The diagnostic path can be graphically represented as follows:

Another aspect of the invention relates to methods for determining kidney functionality based on the comparison of the Ca isotope difference in a previously taken urine and blood sample.

It was shown that the Ca isotope difference Δ _{urine blood} = δ ^44/42 Ca _urine - δ ^44/42 Ca _blood reflects the kidney's recycling efficiency for trace elements and that this relationship is non-linear but can be calculated to the extent that the Values the recycling rate for _{renal reabsorption of} the kidney can be calculated. ( . and ).

The following graphic shows the calcium flows and isotope fractionation between the blood system and the kidneys. With the help of calcium isotope ratios measured in blood and urine, the rate of renal Reabsorption of calcium in the kidney can be calculated. The latter is a measure of the functionality of the kidneys.

The functionality of the kidneys can then be classified according to the invention according to Table 1 on the basis of the calculated recycling rate: Table 1: Classification (threshold values) of kidney function by means of renal reabsorption (f _{renal reabsorption} ) 1.00> f _{renal reabsorption} = ≥ 0.98 → very good kidney function 0.98> f _{renal reabsorption} = ≥ 0.95 → good kidney function 0.95> f _{renal reabsorption} = ≥ 0.92 → renal dysfunction 0.92> f _{renal reabsorption} → increasing kidney dysfunction

The values were calibrated by determining the eGFR (Estimated Glomual Filtration Rate), which was carried out on 12 patients with different kidney functions. Regarding the glomerular filtration rate (GFR) it can be said that this is the currently established method for assessing renal function.

The GFR is either determined relatively laboriously from 24-hour urine collection as creatinine clearance or calculated using various approximate formulas eGFR from the plasma creatinine concentration taking into account skin color, age, gender and other sizes.

The so-called MDMR formula [ Saulo Klahr, Andrew S. Levey, Gerald J. Beck, Arlene W. Caggiula, Lawrence Hunsicker, John W. Kusek, Gary Striker, The Modification of Diet in Renal Disease Study Group: The Effects of Dietary Protein Restriction and Blood-Pressure Control on the Progression of Chronic Renal Disease. In: N Engl J Med. 330, No. 13, March 31, 1994, pp. 877-884. doi: 10.1056 / NEJM199403313301301 ].

The approximate formulas have been validated for outpatients with chronic kidney disease with moderate to severe renal impairment (stages 3 and 4). As a rule, the formulas cannot be used for mild kidney dysfunction or no kidney dysfunction.

• • normaler Nierenfunktion
• • leichter Nierenfunktionseinschränkung.
• • Insbesondere die MDRD-Formel unterschätzt bei Menschen mit einer glomerulären Filtrationsrate über 60 ml/min diese um ca. 10 ml/min [ Stevens, Lesley A. et al.: Evaluation of the Modification of Diet in Renal Disease Study Equation in a Large Diverse Population. In:J Am Soc Nephrol. Nr. 18, 2007, S. 2749-2757 (asnjournals.org)]
• • akuter Nierenfunktionsverschlechterung,
• • schwerem Übergewicht
• • stark verminderter Muskelmasse (Amputation von Gliedmaßen, Unterernährung)
• • mit besonders hoher (Nahrungsergänzungen bei Bodybuildern)
• • niedriger Kreatin-Zufuhr mit der Nahrung (Vegetarier).
• • zur Überwachung der Nierenfunktion im besonders wichtigen Frühstadium der diabetischen Nephropathie [Nephrology beyond JASN. Eberhard Ritz Feature Editor: Estimated GFR: Are There Limits to Its Utility? J Am Soc Nephrol, 2006, 17, S. 2077-2085]

The formulas are also not suitable for determining the glomerular filtration rate in people with:

• • normal kidney function
• • slight impairment of kidney function.
• • In particular, the MDRD formula underestimates people with a glomerular filtration rate above 60 ml / min by approx. 10 ml / min [ Stevens, Lesley A. et al .: Evaluation of the Modification of Diet in Renal Disease Study Equation in a Large Diverse Population. In: J Am Soc Nephrol. No. 18, 2007, pp. 2749-2757 (asnjournals.org)]
• Acute deterioration of kidney function,
• • severe overweight
• • greatly reduced muscle mass (amputation of limbs, malnutrition)
• • with particularly high (nutritional supplements for bodybuilders)
• • low creatine intake with food (vegetarian).
• • to monitor kidney function in the particularly important early stage of diabetic nephropathy [Nephrology beyond JASN. Eberhard Ritz Feature Editor: Estimated GFR: Are There Limits to Its Utility? J Am Soc Nephrol, 2006, 17, pp. 2077-2085]

According to the invention, the Ca isotope difference Δ _{urine blood} = δ ^44/42 Ca _urine - δ ^44/42 Ca _blood is used to classify the recycling efficiency of the kidney. The recycling rate for _{renal reabsorption of} the kidney is calculated from the values. The classification is then based on the threshold values from Table 1.

The δ - notation δ ^44/42 Ca can also be transferred to other Ca isotope ratios using the following formula (for example, involving ⁴⁰ Ca, ⁴¹ Ca, ⁴⁶ Ca, ⁴⁸ Ca, ⁴³ Ca): $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

With m3> m2> m1

The parameters δ ^44/42 Ca _blood , δ ^44/42 Ca _urine and F _urine relevant for the calculation are known by measurement. Where F _urine of the daily loss of calcium indicates the urine.

The Ca isotopes and rivers marked with broken lines can be calculated using the Raleigh equations and the isotope balance. The procedure is as follows:

und für Urin $${\delta }^{44/42}C{a}_{Urin}\left(\text{‰}\right)=\left[\frac{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Urin}}{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Standard}}-1\right]$$

The definition of δ notation is: $${\delta }^{44/42}C{a}_{Blut}\left(\text{‰}\right)=\left[\frac{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Blut}}{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Standard}}-1\right]$$

and for urine $${\delta }^{44/42}C{a}_{Urin}\left(\text{‰}\right)=\left[\frac{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Urin}}{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Standard}}-1\right]$$

For simplification, the following is set: $$\begin{array}{ccc}{R}_{Blut}={\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Blut}& \text{and}& R_{Urin}={\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Urin}\end{array}$$

The ratio of R _urine / R _blood then results from equation (03): $$\frac{R_{Urin}}{R_{Blut}}=\frac{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Urin}}{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Blut}}=\frac{{\delta }^{44/42}C{a}_{Urin}-1}{{\delta }^{44/42}C{a}_{Blut}-1}$$

Equation (03) also has the advantage that the ratio of R _urine / R _blood can be calculated without knowing the absolute ( ⁴⁴ Ca / ⁴² Ca) standard value.

The Rayleigh distillation formula is now used to describe the sequestration of a Ca flow and the associated Ca isotope fractionation.

• a) das Verhältnis der gemessenen Ca Isotopenwerte des Urin und des Blutes $${\text{R}}_{\text{Urin}}/{\text{R}}_{\text{Blut}}=\left({\mathrm{\delta }}^{44/42}{\text{Ca}}_{\text{Urin}}-1\right)/\left({\mathrm{\delta }}^{44/42}{\text{Ca}}_{\text{Blut}}-1\right)$$
  
  und folgende unbekannte Größen:
  1. i. Der Verteilungsquotient der die Aufteilung des Ca zwischen Blut und Urin beschreibt (0≤ f_Urin ≤1). Der Ausdruck f_Urin beschreibt dabei den relativen Anteil des mit dem Urin ausgeschiedenen Calciums und f_{renale-Reabsorbtion} = (1-f_Urin) den Anteil des Calciums welcher in den Blutkreis reabsorbiert wird.
  2. ii. der Fraktionierungsfaktor „α“ zwischen Blut und Urin
  3. iii. sowie der Ca Isotopenwert δ^44/42Ca_{renale-Reabsorbtion} des in den Blutkreislauf zurückgeführten Calcium

The following known (measured) quantities are included in the formulas (04) to (06) of the formulas describing the distillation:

• a) the ratio of the measured Ca isotope values of urine and blood $${\text{R}}_{\text{urine}}/{\text{R}}_{\text{blood}}=\left({\mathrm{\delta }}^{44/42}{\text{Ca}}_{\text{urine}}-1\right)/\left({\mathrm{\delta }}^{44/42}{\text{Ca}}_{\text{blood}}-1\right)$$
  
  and the following unknown sizes:
  1. i. The distribution quotient that describes the division of the Ca between blood and urine (0≤ f _urine ≤1). The expression f _urine describes the relative proportion of calcium excreted in the urine and f _{renal reabsorbtion} = (1-f _urine ) the proportion of calcium that is reabsorbed into the bloodstream.
  2. ii. the fractionation factor "α" between blood and urine
  3. iii. and the Ca isotope _value δ ^44/42 Ca _{renal reabsorption} of the calcium returned to the bloodstream

$$\frac{R_{renale-Reabsorbtion}}{R_{Blut}}=\frac{{\delta }^{44/42}C{a}_{renale-Reabsorbtion}-1}{{\delta }^{44/42}C{a}_{Blut}-1}=\frac{\left(1-f_{Urin}{}^{\alpha }\right)}{1-f}$$

$${\delta }^{44/42}C{a}_{Blut}=f_{Urin}\cdot {\delta }^{44/42}C{a}_{Urin}+\left(1-f_{Urin}\right)\cdot {\delta }^{44/42}C{a}_{renale-Reabsorbtion}$$

Equations (04) to (06) are used. $$\frac{R_{Urin}}{R_{Blut}}=f_{Urin}{}^{\left(\alpha -1\right)}$$

$$\frac{R_{renale-ReabsOrbtiOn}}{R_{Blut}}=\frac{{\delta }^{44/42}C{a}_{renale-ReabsOrbtiOn}-1}{{\delta }^{44/42}C{a}_{Blut}-1}=\frac{\left(1-f_{Urin}{}^{\alpha }\right)}{1-f}$$

$${\delta }^{44/42}C{a}_{Blut}=f_{Urin}\cdot {\delta }^{44/42}C{a}_{Urin}+\left(1-f_{Urin}\right)\cdot {\delta }^{44/42}C{a}_{renale-ReabsOrbtiOn}$$

To further simplify and handle equations (04) to (06), the so-called “small approximation” used in mathematics can be used. The advantage of this procedure is that the measured isotopic difference between blood and urine (Δ _{urine blood} = δ ^44/42 Ca _urine - δ ^44/42 Ca _blood ) can be used. Furthermore, a direct relationship between the isotopic urine-blood difference and the filtration efficiency "f" can then be established:

The following applies ("small approximation"): $$\text{ln}\left(\frac{R_{Urin}}{R_{Blut}}\right)\cong \frac{{\delta }^{44/42}C{a}_{Urin}-{\delta }^{44/42}C{a}_{Blut}}{1000}\cong {\Delta }_{Urin-Blut}$$

The small approximation for In (x) = x applies if x << 1. In isotope chemistry, this is used for all areas from approx. -30 to +40% o. Then 1000 In (x) = d ¹ I _a - d ¹ I _b (d ¹ I _a , d ¹ I _b isotope ratios). It must be multiplied by 1000 since the d ¹ I _{a are given} in parts per thousand (‰).

It follows: $$\frac{R_{Urin}}{R_{Blut}}\cong e^{\frac{{\Delta }_{Urin-Blut}}{1000}};$$

Substituting in equation (04) yields: $$\frac{R_{Urin}}{R_{Blut}}=e^{\frac{{\Delta }_{Urin-Blut}}{1000}}=f_{Urin}^{\alpha -1};$$

The procedure for equation (05) is the same, so that the following then applies: $$\frac{R_{renale-ReabsOrbtiOn}}{R_{Blut}}=e^{\frac{{\Delta }_{\left(renale-ReabsOrbtiOn\right)-Blut}}{1000}}=\frac{\left(1-f_{Urin}{}^{\alpha }\right)}{1-f}$$

$$\frac{{\delta }^{44/42}C{a}_{renale-Reabsorbtion}-1}{{\delta }^{44/42}C{a}_{Blut}-1}=\frac{\left(1-f_{Urin}{}^{\alpha }\right)}{1-f}$$

$${\delta }^{44/42}C{a}_{Blut}=f_{Urin}\cdot {\delta }^{44/42}C{a}_{Urin}+\left(1-f_{Urin}\right)\cdot {\delta }^{44/42}C{a}_{renale-Reabsorbtion}$$

The system of equations to be solved is as follows: $$e^{\frac{{\Delta }_{Urin-Blut}}{1000}}=f_{Urin}{}^{\left(\alpha -1\right)}$$

$$\frac{{\delta }^{44/42}C{a}_{renale-ReabsOrbtiOn}-1}{{\delta }^{44/42}C{a}_{Blut}-1}=\frac{\left(1-f_{Urin}{}^{\alpha }\right)}{1-f}$$

$${\delta }^{44/42}C{a}_{Blut}=f_{Urin}\cdot {\delta }^{44/42}C{a}_{Urin}+\left(1-f_{Urin}\right)\cdot {\delta }^{44/42}C{a}_{renale-ReabsOrbtiOn}$$

The transformed non-linear system of equations (09) to (11) is identical to that in (04) to (06), but shows analytically, especially with equation (09), that there is a functional non-linear relationship between the Ca isotope difference between blood and urine and the recycling rate "f" of the kidneys. This system of equations has three unknowns (f, α and δ ^44/42 _{renal reabsorption} ) and can therefore be solved with the three equations available. However, it is a non-linear system, so the rules of linear algebra do not apply here, but iterative optimizations have to be used to find a simultaneous solution.

The function values of equations (04) and (05) or equivalent (09) and (10) can be displayed graphically. (Rayleigh equations)

The upper curve reflects the Ca isotope composition of urine ( ^44/42 Ca _urine ) as a function of the calcium remaining in the urine. The lower curve reflects the Ca isotopy of the renally reabsorbed (δ ^44/42 Ca _{renale reabsorbed} ) and calcium reabsorbed into the bloodstream. The fractionation factor "α" is responsible for the isotopic difference and its quantitative size.

In the the relationship between the isotopic difference between blood and urine (Δ ^44/42 Ca _{blood urine} ) and the rate of renal reabsorption (f _{renal reabsorption} ) is shown graphically. The areas of kidney functionality (see above) are also shown.

In a further aspect of the invention there is a method for determining the individual calcium (Ca) uptake from the intestine into the blood via the difference in the Ca isotope ratios δ ^44/42 Ca (delta notation). If osteoporosis is identified, important information can be given to the treating physician, in particular in connection with the findings from the functionality of the kidneys about the recycling efficiency of the kidney Δ _{urine blood} = δ ^44/42 Ca _urine - δ ^44/42 Ca _blood , which problems could cause osteoporosis and which therapies are advisable.

The determination is made on the basis of the difference in calcium isotope ratios in blood, stool and in food. A total of several samples taken over a period of a few days are used here.

Based on the knowledge gained in the Osteogeo study, the calculation of the absorption rate via the intestinal blood barrier should be explained.

The δ ^44/42 Ca _food value measured in the study is -0.43 ± 0.01% o.

The measured value δ ^44/42 Ca _{chair of} the chair is -0.32 ± 0.09 ‰.

• Aus den drei nicht-linearen Gleichungen und den zwei gemessenen bekannten Isotopenwerten (δ^44/42Ca_Nahrung und δ^44/42Ca_Stuhl) können die drei unbekannten Größen f_Blut-Darm, α, und δ^44/42Ca_absorbiert berechnet werden:

$$\frac{R_{Stuhl}}{R_{Nahrung}}=f_{Blut-Darm}{}^{\left(\alpha -1\right)}$$

$$\frac{R_{absorbiert}}{R_{Nahrung}}=\frac{\left(1-f_{Blut-Darm}{}^{\alpha }\right)}{1-f_{Blut-Darm}}$$

$${\delta }^{44/42}C{a}_{Nahrung}=f_{Blut-Darm}\cdot {\delta }^{44/42}C{a}_{Stuhl}+\left(1-f_{Blut-Darm}\right)\cdot {\delta }^{44/42}C{a}_{absorbiert}$$

An unknown relative amount of calcium (f _{blood-intestine} ) is absorbed by the blood (f _{blood-intestine} : 0≤ f _{blood-intestine} ≤1, where f _{blood-intestine is} called the absorption rate), and a complementary Ca amount (1-f _{Blood-intestine} ) is excreted in the stool. When the Ca passes from the intestine into the blood, an isotope fractionation of unknown size (α) occurs, so that the Ca isotope value of the absorbed Ca (δ ^44/42 Ca _absorbed ) is also unknown. The three unknown quantities f _{blood-intestine} , α, and δ ^44/42 Ca _absorbed can be calculated using three nonlinear equations:

• From the three non-linear equations and the two measured known isotope values (δ ^44/42 Ca _food and δ ^44/42 Ca _stool ) the three unknown quantities f _{blood-intestine} , α, and δ ^44/42 Ca _absorbed can be calculated:

$$\frac{R_{StuHl}}{R_{NaHrunG}}=f_{Blut-Darm}{}^{\left(\alpha -1\right)}$$

$$\frac{R_{absOrbiert}}{R_{NaHrunG}}=\frac{\left(1-f_{Blut-Darm}{}^{\alpha }\right)}{1-f_{Blut-Darm}}$$

$${\delta }^{44/42}C{a}_{NaHrunG}=f_{Blut-Darm}\cdot {\delta }^{44/42}C{a}_{StuHl}+\left(1-f_{Blut-Darm}\right)\cdot {\delta }^{44/42}C{a}_{absOrbiert}$$

"R" is the measured ⁴⁴ Ca / ⁴² Ca ratio, which can be calculated back from the δ notation:

The following applies: $$\frac{{\delta }^{44/42}C{a}_{StuHl}}{{\delta }^{44/42}C{a}_{NaHrunG}}=\frac{R_{StuHl}-1}{R_{NaHrunG}-1}\text{and}\frac{{\delta }^{44/42}C{a}_{absOrbiert}}{{\delta }^{44/42}C{a}_{NaHrunG}}=\frac{R_{StuHl}-1}{R_{NaHrunG}-1}$$

However, it is a non-linear system, so the rules of linear algebra do not apply here, but iterative optimizations have to be used to find a simultaneous solution. The average absorption rate for _{blood-intestine} is, according to information from the medical literature [Heaney RP. The Calcium Economy. p145-162, In: Weaver CM, Heaney RP, editors. Calcium in Human Health. Totowa, New Jersey: Human Press; 2006] with f _{blood-intestine} = 0.3 thus 30%. In the cited literature reference it is shown that a test person who consumes 20 mmol / d (0.8 g _Ca / day) with food and 3.4 mmol / d (0.136 g _Ca / day) from the Ca-containing digestive juices, 16.6 mmol / d ( 0.66 gca / day). If one only takes food into account, the intake is 17%, if one takes food and the Ca-containing digestive juices into account, the intake is approx. 30%.

A value of f _{blood-intestine} = 0.3, i.e. 30% - taking into account the Ca-containing digestive juices - can therefore be regarded as the target value with an average Ca intake of approx. 1 g calcium per day.

The δ - notation δ ^44/42 Ca can also be transferred to other Ca isotope ratios using the following formula, as in the previous inventions (for example with the participation of ⁴⁰ Ca, ⁴¹ Ca, ⁴⁶ Ca, ⁴⁸ Ca, ⁴³ Ca): $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

With m3> m2> m1

As a result, the doctor receives the knowledge whether the present osteoporosis was triggered by a renal osteopathy (renal dysfunction) or by a reduced calcium (Ca) absorption from the intestine, and thus secondary osteoporosis is present.

Material and methods:

The exemplary procedure is explained below, without restricting the generality of the teaching.

Blood and urine sampling:

The doctor takes a blood sample from the patient with a normal amount of blood (approx. 8 ml) required for blood tests. The blood sample is left to stand for half an hour after which it is centrifuged. The blood serum thus created is separated from the blood cake. Only blood serum is used for the further chemical preparations.

For the further chemical processing, a blood amount is taken which corresponds to an absolute amount of 50 µg calcium.

The patient can take the urine sample, approx. 10 ml, using a container provided for this purpose. For further chemical processing, an amount of urine is taken, which, like blood, corresponds to an absolute amount of 50 µg calcium.

Extraction of the alkaline earth elements from the samples:

The calcium is extracted from the blood and urine using a chemical process until a solution with a concentration of approx. 5 ppm is available for mass spectrometric measurement.

The blood can be prepared for the chemical extraction of calcium using the following procedure:

Chemical sample preparation for calcium isotope determination in blood and urine

• day 1
  • • The vessels prescribed for microwave digestion (MW) are filled with HNO ₃ and H ₂ O ₂ .
  • • The prepared samples and standards are pipetted into the respective tube.
  • • Vessels are closed, the microwave oven is started and placed in the MW for 1.5 hours.
  • • The digested samples are taken from the MW. In the fume cupboard, the solutions are poured into beakers one after the other and placed on the heating plate for drying (overnight).
• day 2
  • • The digested and dried overnight samples are taken up with 1 ml HNO ₃ + 0.5 ml H ₂ O ₂ and boiled again for 3 hours.
  • • Open the beaker and dry the solution.
  • • Dried samples are taken up in 1 ml 2M HNO ₃ .
• Day 3
  • • The concentration of the alkaline earth elements strontium (Sr), calcium (Ca and magnesium) is measured on the Q-ICP-MS using standard methods.
• Day 4
  • • In order to always have the same absolute Ca amounts (50 µg _Ca ) for column chromatography, the amount of acid required for the automated measurement on an ESI PrepFast is calculated and the samples are diluted accordingly.
  • • The samples are transferred from the beakers into the PrepFAST ^® tubes (from Elemental Scientific).
  • • The alkaline earth elements are separated automatically using the prepFAST ^® device (from Elemental Scientific).
• Day 5
  • • The samples, separated by elements, are transferred from the tubes back into the beakers and placed on the hot plate to dry.
  • • After drying, they are taken up again with 1 ml HNO ₃ and 0.5 ml H ₂ O ₂ and cooked closed for a further 3 hours.
• Day 6
  • • The beakers with the samples are opened and dried.
  • • The samples are taken up in 10 ml HNO ₃ and left for 4 hours for equilibration.
  • • The samples are transferred to the tubes for mass spectrometric measurement on the Neptune ^® (plasma mass spectrometer, from ThermoFisher) and measured there.

To determine the isotope ratios and / or ratios of alkaline earth elements, besides the mass spectrometric methods, spectroscopic methods can also be used which use the mass dependence of the hyperfine structure of the spectral lines by means of light emission. The general validity of the method is described in the following, the determination of the values by mass spectroscopy.

Mass spectrometric measurement:

This solution is usually measured in a plasma mass spectrometer (MC-ICP-MS: Multi-Collector-Ionization Coupled - Mass spectrometer) (e.g. ThermoFisher, Neptune) (This procedure is briefly described below), but can also be used as an alternative be measured in a thermionic mass spectrometer. The goal is to determine the calcium isotope composition in blood serum and / or urine. ⁴⁴ Ca / ⁴⁰ Ca ratios are measured in the TIMS and ⁴⁴ Ca / ⁴² Ca or alternative ratios in the MC-ICP-MS. Both isotope ratios are equivalent in their statement. The values differ only by one factor: ⁴⁴ Ca / ⁴⁰ Ca ≅ 2.05 · ⁴⁴ Ca / ⁴² Ca.

Determination of the calcium isotope composition ⁴⁴ Ca / ⁴² Ca using a plasma mass spectrometer:

Calcium isotope measurements are performed on a MC-ICPMS (Neptune ^®, Thermo Fisher Scientific). The mass spectrometer is equipped with nine Faraday beakers, eight of which are mobile. These are set up so that the atomic masses (u) 42, 43, 43.5 and 44 can be measured simultaneously. To suppress interfering Ca and Ar hydrides (e.g. ⁴⁰ Ar ₁ H ₂ to ⁴² Ca), an APEX IR (Elemental Scientific ^® ) sample introduction system is used. All measurements are carried out in medium resolution (m / Δm ~ 4000) on the interference-free plateau on the low-mass side of the central measuring peak. This is achieved by selecting a suitable mean cup mass of 4,687 ± 0.001 u and verifying it daily.

The instrumental fractionation ("mass bias") is corrected using the "Standard Sample Bracketing, (SSB)" procedure. The measurement of a sample is corrected by measuring a 5 µg / ml Ca solution made from a 10000 µg / ml Ca ICP standard solution. Each sample is measured at least four times during a session and its average value is used for the further procedure. The Ca isotope composition is given as δ ^44/42 Ca in parts per thousand (% o): $${\mathrm{\delta }}^{44/42}\text{Ca}\left(\text{‰}\right)=\left[{\left({}^{44}\text{Ca}{/}^{42}\text{Ca}\right)}_{\text{sample}}/{\left({}^{44}\text{Ca}{/}^{42}\text{Ca}\right)}_{\text{reference}}\right]-1$$

• δ^44/42Ca_SRM915a (Probe, %o) = δ^44/42Ca_ICP (Probe) - δ^44/42Ca_ICP (SRM 915a), d.h. alle Messwerte werden relative zum internationalen Standard SRM915a angegeben.

The measured Ca-ICP standard solution serves as the primary reference material. The δ ^44/42 Ca _ICP values are then converted from NIST SRM 915a to NIST SRM 915a using the measured δ ^44/42 Ca _ICP value:

• δ ^44/42 Ca _SRM915a (sample,% o) = δ ^44/42 Ca _ICP (sample) - δ ^44/42 Ca _ICP (SRM 915a), ie all measured values are given relative to the international standard SRM915a.

During each session, chemically unprocessed NIST SRM 915a material is measured at the beginning and end of each session and these results compared to those of chemically processed NIST SRM 915a. The average difference between processed and unprocessed SRM 915a should generally be less than 0.01 ‰, so that the Ca isotope fractionation during chemical cleaning can be viewed negligibly.

The "on-peak" approach is used for background correction. The measured intensities of a 1% HNO ₃ solution are subtracted from the intensities of the subsequent sample measurements.

In addition, the measurements are checked for residues of doubly charged strontium ( ⁸⁴ Sr, ⁸⁶ Sr and ⁸⁸ Sr) in order to ensure the correct measurement of the intensities of the masses of ⁴² Ca, ⁴³ Ca and ⁴⁴ Ca. For this purpose, the intensity ratios of the masses 42 / 43.5, 43 / 43.5 and 44 / 43.5 of a 2 µg / ml Sr solution are measured at the beginning of each session. These ratios are then used to calculate the intensities of doubly charged Sr from the measured mass intensities 43.5 of a given sample.

For quality assurance, the δ ^44/42 Ca values of NIST SRM 1486 and IAPSO seawater standard measured during the measurement phase are compared with published values. Long-term reproducibility (2 SD = standard deviation) over a period of about two months is generally better than 0.06% o for all analyzed reference materials.

• • | δ^44/42Ca - 2·δ^43/42Ca | >0.2 ‰ gilt.
• • Eine Probenmessung wird verworfen, wenn die durchschnittliche Intensität außerhalb eines Intensitätsfensters von 70 bis 130 % liegt, verglichen mit der durchschnittlichen Intensität der 5µg/ml Ca ICP -Lösung oder NIST SRM 915a-Lösung derselben Charge.
• • Eine vollständige Sequenz wird abgelehnt, wenn mehr als eines der gemessenen internationalen Referenzmaterialien mehr als 0.2 %o vom Literaturwert entfernt ist oder die Daten nicht entlang der massenabhängigen Fraktionierungslinie fielen.

For further quality assurance, several criteria are used to reject data from a single measurement, a single sample and entire sequences. A single measurement or sequence is discarded if:

• • | δ ^44/42 Ca - 2 δ ^43/42 Ca | > 0.2 ‰ applies.
• • A sample measurement is discarded if the average intensity is outside an intensity window of 70 to 130% compared to the average intensity of the 5µg / ml Ca ICP solution or NIST SRM 915a solution from the same batch.
• • A complete sequence is rejected if more than one of the measured international reference materials is more than 0.2% o of the literature value or the data did not fall along the mass-dependent fractionation line.

The Ca / Sr ratio is determined using a standard method on a quadrupole mass spectrometer (the method is described below).

Determination of the Ca / Sr and Mg / Ca element ratios

Before the Ca isotope analysis, the Ca, Mg and Sr concentrations of the sample solutions are measured on a quadrupole mass spectrometer (Agilent 7500cx ^® ) using a matrix-adapted external calibration curve and indium as an internal standard. During the measurement, the intensity of the double-charged ions is kept below 2% in order to minimize interference with double-charged ⁸⁸ Sr, ⁸⁶ Sr and ⁸⁴ Sr at ⁴⁴ Ca, ⁴³ Ca and ⁴² Ca. The total blank of the solution is usually less than 50 ng. The long-term reproducibility of the concentration measurements and their relationships using internationally available standards is usually better than 5% (1 SD = standard deviation).

Evaluation of the calcium isotope data:

The measured calcium isotope values are reported in the usual δ notation: $${\delta }^{44/42}Ca=\left[\frac{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{PrObe}}{{\left(\frac{{}^{44}Ca}{{}^{42}Ca}\right)}_{Standard}}-1\right]\cdot 1000$$

Where ( ⁴⁴ Ca / ⁴² Ca) _{sample is} the ratio of the blood sample measured in the mass spectrometer and ( ⁴⁴ Ca / ⁴² Ca) _{standard is} the calcium isotope ratio of an internationally known and certified ratio. This approach allows the measured isotope ratios to be compared internationally.

Determination of the threshold values:

In a clinical study (Osteogeo study), statistically significant threshold values for the differentiation of women for δ ^44/42 Ca _blood , δ ^44/42 Ca _urine and (Ca / Sr) _urine within the framework of ROC (Receiver Operating Characteristics) Analyzes determined. In order to meet the definition of osteoporosis in accordance with the applicable international (WHO World Health Organization) and national (DVO umbrella organization for osteoporosis), the study participants had to exclude those who showed a vitamin D deficiency (≤ 25 µmol / l). This affected 20 women, leaving the study itself with 80 women who met the standard criteria for osteoporosis. Of these 80 women, 66 were characterized as having no osteoporosis using the X-ray findings (DXA, golden standard) and 14 were diagnosed as having osteoporosis. Table 2: ROC determined threshold values: With vitamin Threshold sensitivity Specificity L ^- ; More negative L ⁺ ; More positive D shortage * (%O) (%) (%) Likelihood factor Likelihood factor δ ^44/42 Ca _blood -0.85 ± 0.06 100.0 55 0 2.2 (1.69 - 2.87) δ ^44/42 Ca _urine 0.16 ± 0.06 78.6 71.2 0.3 (0.11 - 0.83) 2.73 (1.71- 4.36) [Ca / Sr] _urine 2027 ± 250 63.3 75.8 0.47 (0.23 - 0.97) 2.65 (1.49 - 4.73) All Threshold sensitivity Specificity L ^- ; More negative L ⁺ ; More positive (%O) (%) (%) Likelihood factor Likelihood factor δ ^44/42 Ca _blood -0.85 ± 0.06 94.4 53.7 0.10 (0.02 - 0.70) 2.04 (1.57 - 2.64) δ ^44/42 Ca _urine 0.23 ± 0.06 72.2 67.1 0.41 (0.19 - 0.89) 2.19 (1.44 - 3.34) [Ca / Sr] _urine 1772 ± 250 72.2 61.0 0.46 (0.21 - 0.98) 1.85 (1.25 - 2.75) Note: * Vitamin D (≤ 25 µmol / l).

The negative likelihood factor (L ^- ) gives the probability of a value above the threshold to get a positive osteoporosis finding relative to a healthy person.

The positive likelihood factor (L ⁺ ) gives the probability of a value below the threshold to get a positive osteoporosis finding relative to a healthy person.

Exemplary procedure in the diagnosis according to the invention using a blood (δ ^44/42 Ca _blood ) and urine sample (δ ^44/42 Ca _blood )

In view of the great similarity of the values (Table 2), all 100 subjects in the Osteogeo study were considered without being restricted by the vitamin D concentration. Nevertheless, the sensitivity of the diagnosis increases if the vitamin D concentration is known (Table 2).

The statistical ROC analysis revealed a threshold value for δ ^44/42 Ca _blood of -0.85 ± 0.06 ‰ (δ ^44/42 Ca _{threshold blood} ) for the entire study group. The sensitivity of the method was calculated to be 94.4% and the specificity is 53.7% (Table 2). Values of healthy subjects measured in the blood above this threshold value are only 0.1 times more likely to get a positive result (osteoporosis) (negative likelihood factor L ^- = 0.1). Measured calcium isotope values below the threshold value are 2.04 times more likely that a sick person (positive likelihood factor L ⁺ = 2.04) will get a positive result (osteoporosis).

Exemplary procedure for the osteoporosis / kidney function diagnosis according to the invention in the event that both the calcium isotope value of a blood sample (δ ^44/42 Ca _blood ) and a urine sample (δ ^44/42 Ca _urine ) are available - Figure 8:

1. a) Form the difference Δ _{urine blood} and calculate the renal reabsorption (f _{renal reabsorption} ). Classification of kidney function according to Table 1.
2. b) If δ ^44/42 Ca _{blood measured} ≥ δ ^44/42 Ca _{threshold blood} + 0.06. Values of healthy subjects measured in the blood which are above this threshold value including the tolerance value are only 0.1 times more likely to get a positive osteoporosis result. The trafficlight is green! There is a high probability that there is no osteoporosis (traffic light is green!)
3. c) If δ ^44/42 Ca _{threshold blood} + 0.06 ≥ δ ^44/42 Ca _{blood measured} ≥ δ ^44/4 Ca _{threshold blood} - -0.06 ‰. Due to the situation within the tolerance range of the threshold value, the patient is diagnosed with an onset of osteoporosis. The traffic light is yellow! These patients will receive an additional DXA measurement Recommended to determine the individual risk of a broken bone for specific bones (eg thighs).
4. d) If δ ^44/42 Ca _{blood measured} ≤ δ ^44/42 Ca _{threshold blood} -0.06 ‰. Calcium isotope values measured in the blood below the threshold value, including the tolerance value, are 2.04 times more likely to have a positive result (osteoporosis).

There is probably a net loss of calcium and osteoporosis! An additional DXA measurement is recommended for these patients in order to determine the individual risk of a broken bone for specific bones.

Below is a graphical representation of the diagnostic path:

Exemplary procedure in the diagnosis according to the invention in the event that only one blood sample (δ ^44/42 Ca _blood ) is available:

1. a) Gilt δ^44/42Ca_{Blut-gemessen} ≥ δ^44/42Ca_{Schwellenwert-Blut} + 0.06:
   • Gemessene Calcium Isotopenwerte oberhalb dieses Schwellenwertes einschließlich der Toleranz haben nur eine 0.1 fache Wahrscheinlichkeit einen positiven Befund (Osteoporose) zu bekommen (Die Ampel ist grün!).
2. b) Gilt δ^44/42Ca_{Schwellenwert-Blut} + 0.06: ≥ δ^44/42Ca_{Blut-gemessen} ≥ δ^44/42Ca_{Schwellenwert-Blut} - 0.06:
   • Aufgrund der Lage innerhalb des Toleranzbereichs des Schwellenwertes wird dem Patient eine beginnende Osteoporose diagnostiziert (Die Ampel ist gelb!)! Diesen Patientinnen wird eine zusätzliche DXA-Messung empfohlen um für spezifische Knochen das individuelle Risiko für einen Knochenbruch zu bestimmen.
3. c) Gilt δ^44/42Ca_{Blut-gemessen} ≤ δ^44/42Ca_{Schwellenwert-Blut}- 0.06:
   • Gemessene Ca Isotopenwerte unterhalb des Schwellenwertes einschließlich der Toleranz haben eine 2.04 fach höhere Wahrscheinlichkeit einen positiven Befund (Osteoporose) zu bekommen (Die Ampel ist rot!). Es besteht der Verdacht auf einen allgemeinen Calcium Verlust und mögliche Osteoporose. Diesen Patientinnen wird eine zusätzliche DXA-Messung empfohlen um für spezifische Knochen das individuelle Risiko für einen Knochenbruch zu bestimmen.
     

A statement about osteoporosis can also be made if only one blood sample is available. Due to the higher sensitivity of the blood samples compared to the urine sample (Table 2), the diagnostic information of a blood sample is higher than that of a urine sample, since the urine sample is restricted by a Kidney function may be affected. The sensitivity of the diagnostic statement increases further if the patient's vitamin D concentration is known (Table 2).

1. a) If δ ^44/42 Ca _{blood measured} ≥ δ ^44/42 Ca _{threshold value blood} + 0.06:
   • Measured calcium isotope values above this threshold, including the tolerance, are only 0.1 times more likely to get a positive result (osteoporosis) (the traffic light is green!).
2. b) If δ ^44/42 Ca _{threshold blood} + 0.06: ≥ δ ^44/42 Ca _{blood measured} ≥ δ ^44/42 Ca _{threshold blood} - 0.06:
   • Due to the situation within the tolerance range of the threshold value, the patient is diagnosed with osteoporosis (the traffic light is yellow!)! An additional DXA measurement is recommended for these patients in order to determine the individual risk of a broken bone for specific bones.
3. c) If δ ^44/42 Ca _{blood measured} ≤ δ ^44/42 Ca _{threshold blood} - 0.06:
   • Measured Ca isotope values below the threshold including tolerance are 2.04 times more likely to get a positive result (osteoporosis) (the traffic light is red!). There is a suspicion of a general loss of calcium and possible osteoporosis. An additional DXA measurement is recommended for these patients in order to determine the individual risk of a broken bone for specific bones.
     

Exemplary procedure for the diagnosis according to the invention in the event that only one urine sample (δ ^44/42 Ca _urine ) is available:

1. a) Gilt δ^44/42Ca_{Urin-gemessen} ≥ δ^44/42Ca_{Schwellenwert-Urin} + 0.06:
   • Gemessene Calcium Isotopenwerte oberhalb dieses Schwellenwertes einschließlich der Toleranz haben nur eine 0.41 fache Wahrscheinlichkeit einen positiven Befund (Osteoporose) zu bekommen (Die Ampel ist grün!).
2. b) Gilt δ^44/42Ca_{Schwellenwert-Urin} + 0.06: ≥ δ^44/42Ca_{Urin-gemessen} ≥ δ^44/42Ca_{Schwellenwert-Urin} - 0.06:
   • Aufgrund der Lage innerhalb des Toleranzbereichs des Schwellenwertes wird dem Patient eine beginnende Osteoporose diagnostiziert (Die Ampel ist gelb!)! Diesen Patientinnen wird eine zusätzliche DXA-Messung empfohlen um für spezifische Knochen das individuelle Risiko für einen Knochenbruch zu bestimmen.
3. c) Gilt δ^44/42Ca_{Urin-gemessen} ≤ δ^44/42Ca_{Schwellenwert-Urin}- 0.06:
   • Gemessene Ca Isotopenwerte unterhalb des Schwellenwertes einschließlich der Toleranz haben eine 2.19 fach höhere Wahrscheinlichkeit einen positiven Befund (Osteoporose) zu bekommen (Die Ampel ist rot!). Es besteht der Verdacht auf einen allgemeinen Calcium Verlust und mögliche Osteoporose. Diesen Patientinnen wird eine zusätzliche DXA-Messung empfohlen um für spezifische Knochen das individuelle Risiko für einen Knochenbruch zu bestimmen.
     

• : Calcium Isotopie der Nahrung, des Stuhls, des Blutes und des Urin bei den postmenopausalen Frauen.
• : Zusammenhang zwischen dem isotopischen Unterschied von Blut und Urin (Δ^44/42Ca_Blut-Urin) sowie der Rate der renalen Reabsorbtion (f_{renale-Reabsorbtion}) als direktes Maß für die Nierenfunktion. Zusätzliche sind die definierten Bereiche unterschiedlicher Nierenfunktionen (Schwellenwerte) dargestellt.
  
  
  

A statement about osteoporosis can also be made if only a urine sample is available. The sensitivity of the diagnostic statement increases if the patient's vitamin D concentration is known.

1. a) If δ ^44/42 Ca _{urine measured} ≥ δ ^44/42 Ca _{threshold urine} + 0.06:
   • Measured calcium isotope values above this threshold, including the tolerance, are only 0.41 times more likely to get a positive result (osteoporosis) (the traffic light is green!).
2. b) If δ ^44/42 Ca _{threshold urine} + 0.06: ≥ δ ^44/42 Ca _{urine measured} ≥ δ ^44/42 Ca _{threshold urine} - 0.06:
   • Due to the situation within the tolerance range of the threshold value, the patient is diagnosed with osteoporosis (the traffic light is yellow!)! An additional DXA measurement is recommended for these patients in order to determine the individual risk of a broken bone for specific bones.
3. c) If δ ^44/42 Ca _{urine measured} ≤ δ ^44/42 Ca _{threshold urine} - 0.06:
   • Measured Ca isotope values below the threshold value including the tolerance are 2.19 times more likely to get a positive result (osteoporosis) (the traffic light is red!). There is a suspicion of a general loss of calcium and possible osteoporosis. An additional DXA measurement is recommended for these patients in order to determine the individual risk of a broken bone for specific bones.
     

• : Calcium isotope of food, stool, blood and urine in postmenopausal women.
• : Relationship between the isotopic difference between blood and urine (Δ ^44/42 Ca _{blood urine} ) and the rate of renal reabsorption (f _{renal reabsorption} ) as a direct measure of kidney function. The defined areas of different kidney functions (threshold values) are also shown.
  
  
  

• - eine Verpackung, diese umfassend:
• - einen Auffangbehälter zur Aufnahme der Probenflüssigkeit,
• - mindestens eine Monovette zur Aufnahme eines Teils der Probenflüssigkeit und zum Transport zu einem Messgerät, das Isotopenverhältnisse und/oder Mengenverhältnisse von Erdalkalimetall-Anteilen bestimmt, und
• - eine Anleitung,

wobei die Anleitung einen festen vom Individuum unabhängigen Schwellenwert für das Isotopenverhältnis und/oder das Mengenverhältnis der Erdalkalimetall-Anteile zum Vergleich mit dem oder den zu ermittelnden Probenwerten ausweist.The invention further relates to a test kit for use in a method for diagnosing a disease which is associated with a reduced bone density and / or calcium loss, comprising:

• - a package comprising:
• a collecting container for holding the sample liquid,
• - at least one monovette for taking up part of the sample liquid and for transport to a measuring device which determines isotope ratios and / or quantitative ratios of alkaline earth metal fractions, and
• - an instruction,

the instructions show a fixed threshold for the isotope ratio and / or the quantitative ratio of the alkaline earth metal fractions, which is independent of the individual, for comparison with the sample value or samples to be determined.

A fixed threshold value for the isotope ratio ⁴⁴ Ca / ⁴² Ca after a δ notation, in particular of δ ^44/42 Ca _urine = 0.23 ± 0.06% o for a urine test kit or δ ^44/42 Ca _blood = - 0.85 ± 0.06% o for a blood test kit can be identified in the test kit instructions.

• - eine Verpackung, diese umfassend:
• - einen Auffangbehälter zur Aufnahme der Probenflüssigkeit,
• - mindestens eine Monovette zur Aufnahme eines Teils der Probenflüssigkeit und zum Transport zu einem Messgerät, das Isotopenverhältnisse und/oder Mengenverhältnisse von Erdalkalimetall-Anteilen bestimmt, und
• - eine Anleitung, wobei ein von einem oder mehreren durch den individuellen Patienten bedingten Faktoren unabhängiger Schwellenwert zum Vergleich des mindestens einen Probenwertes mit dem Schwellenwert in dem Testkit angegeben ist.

The present invention further relates to a test kit for use in a method according to the invention, comprising

• - a package comprising:
• a collecting container for holding the sample liquid,
• - at least one monovette for taking up part of the sample liquid and for transport to a measuring device which determines isotope ratios and / or quantitative ratios of alkaline earth metal fractions, and
• instructions, wherein a threshold value independent of one or more factors caused by the individual patient for comparing the at least one sample value with the threshold value is specified in the test kit.

Such a test kit can include all of the features mentioned above for the method or can have all the facilities necessary for the method according to the invention and its configurations, individually or in combination.

It is particularly advantageous in the test kit according to the invention that, for the first time, a simple sample is sufficient to provide a basis for a diagnosis relating to a disease which is associated with a reduced bone density and / or calcium loss. A comparison with the fixed threshold value, in particular independent of the patient, represents a very large simplification of the processes of the previously known diagnostic kits.

QUOTES INCLUDE IN THE DESCRIPTION

This list of documents listed by the applicant has been generated automatically and is only included for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent literature cited

• US 2013/0115650 A1 [0009]
• US 2014/0273248 A1 [0011]

Non-patent literature cited

• Skulan, Joseph; et al; "Natural Calcium Isotopic Composition of Urine as a Marker of Bone Mineral Balance" Clinical Chemistry, 53, 1155-1158 (2007) [0010]
• Saulo Klahr, Andrew S. Levey, Gerald J. Beck, Arlene W. Caggiula, Lawrence Hunsicker, John W. Kusek, Gary Striker, The Modification of Diet in Renal Disease Study Group: The Effects of Dietary Protein Restriction and Blood-Pressure Control on the Progression of Chronic Renal Disease. In: N Engl J Med. 330, No. 13, March 31, 1994, pp. 877-884. doi: 10.1056 / NEJM199403313301301 [0069]
• Stevens, Lesley A. et al .: Evaluation of the Modification of Diet in Renal Disease Study Equation in a Large Diverse Population. In: J Am Soc Nephrol. No. 18, 2007, pp. 2749-2757 [0071]

Claims (18)

A method for diagnosing a disease which is associated with a reduced bone density and / or calcium loss, comprising the following steps: a) determining isotope ratios or quantitative ratios of alkaline earth metal fractions in a sample of urine or blood or stool, b) thereby determining at least one sample value , and c) comparison of the at least one sample value with a threshold value, characterized in that the threshold value is selected independently of one or more factors caused by the individual patient. Procedure according to Claim 1 , characterized in that in step a) the isotope ratios or quantitative ratios of the alkaline earth metal fractions in the sample are determined by mass spectrometry. Procedure according to Claim 1 or 2 , characterized in that the isotope ratios of calcium (Ca) are determined in step a). Method according to one of the preceding claims, characterized in that in step a) the quantitative ratio of the alkaline earth elements of calcium (Ca) to strontium (Sr) is determined. Method according to one of the preceding claims, characterized in that in step c) a fixed threshold value which is independent of the individual and which was previously determined depending on the type of sample, serves as a comparison value. Method according to one of the preceding claims, characterized in that the threshold value in step c) for the isotope ratio ⁴⁴ Ca / ⁴² Ca in a blood sample after a δ notation is δ ^44/42 Ca _blood = -0.85 ± 0.06 ‰. Method according to one of the preceding claims, characterized in that the threshold value in step c) for the isotope ratio ⁴⁴ Ca / ⁴² Ca in a urine sample after a δ notation is δ ^44/42 Ca _urine = 0.23 ± 0.06 ‰. Procedure according to Claim 6 , characterized in that the threshold value according to the formula: $${\mathrm{\delta }}^{m3/m2}Ca={\mathrm{\delta }}^{m3/m1}Ca\times \left[\frac{ln\left(m3/m2\right)}{ln\left(m3/m1\right)}\right]$$

with m3>m2> m1, whereby m3, m2, m1 represent the mass numbers, can be transferred to other isotope ratios of calcium. Method according to one of the preceding claims, characterized in that the threshold value in step c) for the quantitative ratio of the alkaline earth elements of calcium (Ca) to strontium (Sr) is 1772 ± 250 mol _Ca / mol _Sr. Method according to one of the preceding claims, characterized in that a sample value as δ ^44/42 Ca _urine and a further sample value as δ ^44/42 Ca _{blood are} determined in step b) and in step c) the difference between the two sample values with a threshold value is compared to make a statement about kidney function. Method according to one of the preceding claims, characterized in that in step b) a sample value as δ ^44/42 Ca _stool and a further sample value as δ ^44/42 Ca _{blood are} determined and in step c) the difference between the two sample values with a threshold value is compared to additionally make a statement about the intestinal function. Method according to one of the preceding claims, characterized in that the disease associated with a reduced bone density and / or calcium loss is osteoporosis, osteomalacia, multiple myeloma and / or renal dysfunction. Method according to one of the preceding claims, characterized in that the quantitative ratios in step a) are determined as molar ratios of the alkaline earth metal fractions in the sample or as mass ratios of the alkaline earth metal fractions in the sample. Method according to one of the preceding claims, characterized in that when in step b) a sample value is determined as δ ^44/42 Ca _urine and in step c) the comparison with the threshold value after a δ notation of δ ^44/42 Ca _urine = 0.23 ± 0.06% o indicates that the sample value is larger, the presence of an osteoporosis disease can be excluded as unlikely. Method according to one of the preceding Claims 1 - 13 , characterized in that if in step b) a sample value is determined as δ ^44/42 Ca _blood and in step c) the comparison with the threshold value after a δ notation of δ ^44/42 Ca _blood = -0.85 ± 0.06% o shows that the sample value is larger, the presence of an osteoporosis disease can be excluded as unlikely. A test kit for use in a method of diagnosing a disease associated with decreased bone density and / or calcium loss, comprising: - a package comprising: - a collection container for holding the sample liquid, - at least one monovette for holding a part of the sample liquid and for transport to a measuring device which determines isotope ratios and / or quantitative ratios of alkaline earth metal fractions, and - a manual, characterized in that the manual has a fixed threshold value for the isotope ratio and / or the quantitative ratio of the alkaline earth metal fractions for comparison with the individual identifies the sample value or values to be determined. Test kit after Claim 16 , characterized in that a threshold value for the isotope ratio 44Ca / 42Ca is shown after a δ notation of 644 / 42CaUrin = 0.23 ± 0.06% o or 644 / 42CaBlut = -0.85 ± 0.06% o. Test kit for use in a method according to one of the Claims 1 to 15 comprising - a packaging comprising: - a collecting container for holding the sample liquid, - at least one monovette for holding a part of the sample liquid and for transport to a measuring device which determines isotope ratios and / or quantitative ratios of alkaline earth metal fractions, and - instructions , wherein a threshold value independent of one or more factors dependent on the individual patient for comparing the at least one sample value with the threshold value is specified in the test kit.