DE3224641A1 - Method for the determination of glomerular filtration rates - Google Patents

Abstract

The invention deals with a method for the determination of the glomerular filtration rate after a single injection of a test dose of a corrinoid and with a method for the determination of tiny amounts of vitamin B12. This may be of importance, for example, in a wide variety of disciplines. An example of this from medicine. Clearance investigations for clinical testing of kidney function are known. Radioactive isotopes are used for this, thus exposing the human body to radioactivity. If it is possible to obtain at least equally good results in clearance investigations without exposing the human body to radioactive isotopes, this would be a great success. This is possible by detection of tiny amounts of vitamin B12 in the human body. This is achieved by saturating the B12 receptors, administering at least one test dose of vitamin B12, and subsequently determining the plasma concentration by HPLC (High Pressure Liquid Chromatography). <IMAGE>

Description

The invention relates to a method as described in the preamble of claim 1.

The invention is suitable for determining the glomerular! Filtration rate using the falling plasma level method.

The invention is also suitable, for example, for the detection of B ₁₂ (cyanocabalamin) in general and can be used, for example, in clinical areas, in veterinary medicine, nutritional chemistry, environmental protection, botany, biochemistry, etc. In particular, small amounts are to be determined according to the invention.

The invention is illustrated below using an example explained in more detail.

For example, clearance studies are considered routine methods for clinical "functional testing of the kidneys

knows. The glomerular filtration function is inter alia with radioactive isotopes such as 51-chromium-EDTA and 57-Co- / 58-Co-cyanocobalamin examined (Kluthe, R. Oechslen, D. (eds): Current diagnostics of kidney diseases. Thieme Verlag, Stuttgart 1974, pp.88-101). These tests are based on the assumption that the decrease in the measured plasma concentration curve of the injected test substance reflects the glomerular filtration rate of the test substance over time. The clearance (Cl) is the amount Plasma (ml), which is freed from the test substance per unit of time:

Cl _cor = dose- _xkx ^ o

It means:

; l = clearance, based on a body surface ^cor of 1.73 m2

y = concentration of the test substance at the (extrapohed) Time t = ο -min. *

k = elimination constant of the function j = jo .e-k.t

Κ. = Correction factor for normalizing to a Body surface of 1.73 m2.

(For formula see Dost, F.H .: Fundamentals of Pharmacokinetics. Thieme Verlag, Stuttgart 1966).

Is disadvantageous; that the human body is contaminated with radioactive isotopes.

The object of the invention is a method for determining small amounts of (for example vitamin B ₂ ) glomerular piltration rates of a corrinoid, for example a method in which radioactive contamination of the human body is avoided, the results preferably being better.

The problem posed is achieved by the characterizing features of claim 1 and those of the subclaims.

If one stays with the example given at the beginning, then no radioactively labeled vitamin B ^ _{2 is} injected, but native, unlabelled vitamin B ₁₂ , which is an endogenous substance anyway. This also eliminates potential damage to the body by exogenous substances (such as 51-chromium EDTA and inulin etc.) or radioactive substances (such as 57- or SS-co-cyanacobalamin). This method has become possible in that further comprises a method for measuring minute quantities of unlabeled vitamin B was invented _12th This method is based on the technique of HPLC «High Pressure Liquid Chromatography.

The procedure for measuring the glomerular filtration rate consists of

«! •• It ♦ · t I

1. a pre-injection to saturate the protein receptors for vitamin B ₁₂ *

2. the actual test injection,

3. taking e.g. five blood samples,

4. Analyze the vitamin B. Concentration in the Blood samples by HPLC and

5. the calculation of the clearance using the above formula.

The procedure can be carried out in detail as follows:

1. 2 to 20 ml, preferably 3 ml of blood (I. blank sample) in a test tube with preferably 50 mg Na-EDTA give, shake, centrifuge, pipette off plasma. I.

2. 1,000 to 30,000, preferably 2,000 ug of cyanocobalamin!

(e.g. vitamin B ₁₂ , MERCK, Darmstadt) inject intravenously. !

3. After 0.5 to 20 hours 3 ml of blood (II. Blank sample) remove.

4. Inject 1,000 to 30,000 µg intravenously.

5. 60 minutes after the injection, preferably after 60; Take 90, 120 minutes, 1 to 10, preferably 3 ml of blood j.

6. When setting up the calibration curve with the HPLC, one ml of cyanocobalamin-free blood plasma becomes a certain Amount of cyanocobalamin added.

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7. In each case 1 ml of plasma with 0.5 nil trichloro acetic acid Add stirring (TCA = 3 mol / l), fill up with 5 ml v / water.

8. Centrifuge. Decant the supernatant. Be careful, that no protein particles are decanted, if necessary decant again.

9. Add 200 ng of bromocresol green to the clear solution.

10. Solution with a Fortuna pipette via the reversed phase Press material (reversed-phase Cjg cartridge before the activate first use with a methanol rinse).

11. Successively with 5 ml water, 1 ml 0.05% NaCN solution. Rinse 3 ml v / ater.

12. Elute adsorbed substances with 1 ml of methanol. 1 3. Take up eluate in Eppendorf reaction tube.

14. Evaporate the solution under a stream of nitrogen at approx. 40 ° C,

15. Take up residue with 8.0 ill 0.1% NaCN solution, centrifuge.,

S. -z-

16. After 10 rain solution, inject into the chromatograph. Measure.

Calculation of the cyanocobalamin concentration: This results from the ratio of the two areas below the peaks for cyanocobalamin and the internal standard (bromocresol green) (see Fig. 1). It is calculated according to the method; Height of the peak times the width of the peak at half height.

Calculation of the glomerular filtration rate:

Π = D .k.Kp,

"" ¹ COr γ -P '

^y ο

where Cl: clearance, corrected for body surface ^cor area of 1.73 m2 (ml / min)

D: Dose, i.e. the amount of cyanocoba injected

lamin (ug)

y: virtual concentration of cyanocobalamin

in the plasma at time t (ng / ml)

k: elimination constant (l / min)

Correction factor J k or ρ er ob er f 1 ach e

K _c : correction factor for 1.73 m ² as standard

= 1.73

Body surface

Body surface area (m ² ) =

= Cfevicht (kp) ⁰ ' ⁴²⁵ χ Height (cm) ⁰ ' ^{725 χ 71.84}

10,000

Instead of the graphic. Extrapolation for determination of y at time t one can also do a linear regression analysis perform (Fig. 4).

—Kt
One takes y = y _Q .e and transforms it into In y = In y - kt.

The cyanocobalamin concentrations of the various blood samples are then taken to calculate In y and k.

For the glomerular filtration rate "were among the The circumstances described here obtained values between 120.5 and 175 ml / min for 5 patients with no detectable Disease (see table). Thus, this method delivers values that are very similar to the inulin clearance, which with *

Method Notes:

Calibration: The standard curve for the correlation between cyanocobalamin concentration in plasma and the relative jagged area in the chromatogram was created by pooling certain amounts of cyanocobalamin in Subject plasma were given. One took a constant amount of bromeresol green in the respective cyanocobalamin-containing Plasma samples as internal standards, the calibration curve ran straight between 20 and 500 ng / ml cyanocobalamin per ml plasma (Fig. 3).

* The help of a constant plasma level of inulin through a continuous infusion is obtained.

Recovery rate:

The relative recovery rate was calculated by comparing the relative jagged area of a 300 ηg / ml cyanocobalamin solution in plasma with that of a 300 ng / ml cyanocobalamin solution in water. Both solutions contained 200 ng / ml bromocresol green. The relative recovery rate was 65.3 + 3.0 % for η = 12.

Precision:

The precision of the measuring process within a day was determined on plasma samples containing certain amounts had added cyanocobalamin. The coefficients of variation were 3.98% for a 500 ng / ml and 6.94% for a 200 ng / ml plasma cyanocobalamin concentration.

Stability:

The high stability of cyanocobalamin in the plasna when stored in the cold or frozen has already been described (Kubasik, Graham, Sine: storage and stability of folate and vitamin B 12 in plasma and blood samples. Clin. Chim. Acta 95. ' 147 to 149, 1979).

Enrichment process:

The separation of the cyanocobalamin from other plasma factors is achieved by its specific accumulation on octadecylsilylated silica (ie the "Reversed Phase · ¹ C ₁₈ cartridge from WATERS). After washing the

-VQ-

Cartridge (= cartridge) is cyanocobalamin through cyanide converted into dicyanocobalamin. Dicyanocobalamin is more stable and has a higher absorption coefficient at 368 nm as cyanocobalamin (see Fig. 2). Dicyanocobalanine is then washed out of the cartridge with methanol, Ss is stabilized by adding cyanide to the PHLC washout agent. For cleaning the column and the cartridge a syringe must be used that does not contain brass parts, even if they are chrome-plated, because Cyanocobalamin is extremely unstable when the central metal ion is exchanged in an acidic medium (Friedrich, V. Vitamin B 12 and related corrinoids. Thieme Verlag, Stuttgart 1975).

HPLC separation:

Examples of HPLC chromatograms for the determination of cyanocobalamin in plasma at certain times according to Injection are shown in FIG.

- rf -

Materials:

A high pressure liquid chromatograph from Knauer, Berlin, model 52.00 pump, model RH 17 (100 μg loop) injection system, 250 × 4.6 mm column filled with 5 μm Lichrosorb SI 60 (E. Merck, Darmstadt), and model 85.00 variable V / e Ilen length detector. The column temperature (25-30 C) was controlled by a model 60.00 oven (Knauer). _{Sep-Pak C 1} g cartridges (Waters, MiIford, USA) were used for the reversed phase cleaning.

Reagents:

Methanol p.a., bromocresol green p.a., NaCN p.a., trichloroacetic acid p.a., Cyanocobalamin p.i (Cytobion ^, 2000 and 5000 µg) were from E. Merck, Darmstadt. Cyanocobalamin as the standard for analysis was from Sigma, Munich.

Solutions:

0.1% NaCN, 3 mol / 1 TCA, 20 mg / l bromocresol green, and 10, 20, 30, 50 mg / l cyanocobalamin; all solutions were prepared with double-distilled water.

Mobile phase:

Methanol - 0.01 % NaCN (40 + 60) at 0.8 ml / min.

Measurement at 368 nm wavelength. - ■

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Determinations of the extracellular space are important in many areas * of internal and surgical medicine, but they are not yet part of routine diagnostics. This is partly because these procedures are lengthy and time consuming and suitable / lacking test substances without harm to the body / inulin is a substance that is broken down by the body very, very slowly and therefore accumulates with repeated use and also causes allergies . The method of determination is also not easy. Other substances such as Na ₀ 35so ^ and bromine salts are radioactively marked as test substances and thus represent a potential radiation risk. The method of determining extracellular space with vitamin _3-2 is completely safe for the body. Even extremely high doses of several 1,000 mg did not lead to toxic symptoms. The preliminary results so far of five healthy test persons are in the range between 18.9 and 24.1 % of the body weight for the extracellular space; the inulin space is nit 16 in the literature up to 22 % for a group older than our test subjects.

The procedure in detail corresponds to that described above. The o .. a. Data for the extra

* s. Tabel. ·.

cellular space (see table, column extracellular space)

were determined at the same time as the glomerular filtration rate data. The volume of distribution for the test substance, in this case vitamin B ₁₂ , is already included in the formula for clearance. This means that two different clinically relevant parameters can be measured and calculated simultaneously with one test.

Tabel

Patiem

3ü 45 60 90
(min)

Vh =
0.57 0.46 0.44 0.34

U, 71 0.60 0.45 0.38
0.56 0.40 0.45 0.36
0.95 0.70 0.65 0.54
0.50 0.47 0.40 U, 2Ü

Uh)

0.72

0.86

0.69

1.1

U, 01

270

322

259

420

315

k
(min " ¹ )

-0.00837

-0.01062

-0.00718

-0.00066

-U, UU11U

xtra-

ellular

aum

(D

5
5

18.5 (22.8%)

15.5 (21.2%)

19.3 (24.1%)

11.9 (18.9%)

1b, 0 (■ 22.6%)

Right now CröOc KO ) (Kp) (cm) 00 81 180 2, 99 73 190 1, 98 00 179 1, 67 63 163 1. 01 70 170

KO factor

0.86

0.87

0, B7

1.03

0.95

Vh = see calibration curve; Serrated area ratio
other symbols: see formula for

Clearance calculation

% = Percent of body weight

(ml / min)

133.3

120.5

106.1

170.9.:,. $

CO K)

CJ)

Claims (3)

Patent claims 1. Method for determining glomerular filtration rates of a corrinoid, characterized in that a test dose - preferably after a single injection - of a corrinoid is administered and subsequently the falling plasma concentrations of this corrinoid with Can be determined using HPLC. 2. The method according to claim 1, characterized in that, to determine small amounts, for example of vitamin B _12, the B ₁₂ receptors are saturated, at least one test dose of B _{1p is} administered and a plasma concentration determination is then carried out with the aid of the PHLC. 3. The method according to claim 1 and / or 2, characterized in that - after the plasma is prepared - bromocresol green to the decanted Plasma added, the solution through a C. «cartridge pushed through the C. g cartridge with Na CN solution flushed through, after evaporation in a stream of nitrogen the residue is centrifuged with Na CN solution and this solution is injected into the chromatograph. *at least