Jun 10, 1991 - 3, 1992 403. Practical Approach for Determining Glomerular Filtration Rate by Single-Injection. Inulin Clearance. Klaus Jung,' Wolfgang Ilenke,' ...
CLIN. CHEM.
38/3, 403-407 (1992)
Practical Approach for Determining Glomerular Filtration Rate by Single-Injection Inulin Clearance Klaus Jung,’ Wolfgang Ilenke,’ Bernd D. Schulze,2 Karin Sydow,3 Klaus Precht,2 We compared the glomerular filtration rate as measured by a single-injection inulin clearance with that measured by a standard isotope method with 9Tc-labeled diethylenetnaminopentaacetic acid in 21 subjects with glomerular filtration rates >35 mL/min. After a bolus injection of 5 g of inulin, blood samples were taken 20, 45, 90, 120, 145, 180, and 240 mm afterwards. Inulin was measured by
optimized chemical or enzymatic methods of high analytical sensitivity to determine inulin at low concentrations. We used the one-compartment model and inulin concentrations measured at two sampling times to calculate the
glomerular filtration rate from the data of the disappearance curve of inulin. Inulin concentrations at 20 and 240 mm after injection of the inulin bolus were suited to estimate glomerular filtration rate by this procedure, resulting in values ( comparable with those obtained by isotope technique (x). The relationship to the isotope technique was characterized by the equation y = +4.80 mLlmin + 0.92x (r = 0.97). The single-injection inulin clearance determination can detect a decrease of glomerular filtration rate at the beginning of kidney damage, given that our study included subjects with glomerular filtration rates >35 mL/min. We conclude that the glomerular filtrationrate can be determined by analyzing only two blood samples after a bolus injection of inulin. Addftlonal Keyphrases: renal function enzymaticmethods
colorimetiy
.
kinetic analysis radioassay compared
For assessing renal function and the rate of progression of renal diseases, measurements of glomerular ifitration rate (GFR) are necessary, both in clinical practice and research (1). However, the standard determination of mum clearance with continuous intravenous infusion and timed collections of urine samples is tedious, impractical, and especially inconvenient for patients. Because conventional methods of determining GFRvia endogenous creatinine clearance are unreliable (2,3) and because the isotope-based techniques require special equipment and have the disadvantages of radiation exposure, clinicians demand practical and accurate alternatives (1,3-5). One possibility is to calculate GFR from the curve for inulin disappearance in plasma after a single injection with inulin (4, 6-B). Two conditions are necessary for the success of this approach. First, one must use methods of high analytical sensitivDepartments of’ Experimental Organ Transplantation,2 Internal Medicine, and 3Nuclear Medicine, Medical Faculty, University Hospital Charite, Humboldt University Berlin, Landaberger Allee 49, D-O 1017 Berlin, F.R.G. Received June 10, 1991; accepted January 10, 1992.
and Silke Klotzek’
inulin at low concentrations, the serum concentrations of mum at the measuring points in this technique being essentially lower than those found with the standard inulin technique (9,10). Second, one must use an appropriate mathematical model to calculate GFR based on the plasma disappearance curve of the injected inulin (6). In a previous paper we described sensitive and reliable assays for measuring low concentrations of inulin in serum (9). These refined methods enabled us to evaluate the diagnostic usefulness of the single-injection inulin clearance. We now have compared the isotope clearance of 9’Tc-labeled diethylenetriaminopentaacetic acid (DTPA) [a good estimate of GFR (5,11,12)1 with values obtained by the single-injection inulin ity to determine
method, using different mathematical approaches. Because we are especially interested in evaluating renal function at the beginning stages of kidney damage, our
study included mtlmin.
only patients
with GFR values
>35
MaterIals and Methods Subjects and Reagents
Twenty-one persons (12 women, nine men; mean age 38 years) having a wide variety of diagnoses-including essential hypertension, diabetes mellitus, interstitial nephritis, and chronic glomerulonephritis-were referred for renal-function evaluation. Their concentrations of serum creatinine were between 61 and 228 pmoL’L (mean value 118 imoJJL). mum was from Laevosan GmbH (Linz, Austria). Anthrone, sulfuric acid, perchioric acid, and trichioroacetic acid were from Laborchemie (Apolda, F.R.G.). All enzymes and biochemicals were obtained from Boehringer Mannheim GmbH (Mannheim, F.R.G.). Other chemicals of analytical grade were from E. Merck (Darmstadt, F.R.G.). #{176}‘‘Tc-DTPA was supplied by Isocommerz GmbH (Berlin, F.R.G.). Inulin Assay
The mum concentration was measured with either an improved enzymatic determination of glucose/fructose or an optimized colorimetric method based on fructose determination with anthrone reagent after acid hydrolysis of inulin (9). Enzymatic method: To 300 ML of serum or water blank add 50 MLof glucose-removing reagent [1.4mg of glucose oxidase (EC 1.1.3.4) and 23 ML of catalase (EC 1.11.1.6)1 in 1 mL of 100 mmol/L triethanolamine . HC1 buffer, pH 7.01 and incubate at 37#{176}C for 4 h. Then add 350 ,uL of 0.54 mmol/L HC1O4, incubate at 80#{176}C for 15 CLINICALCHEMISTRY, Vol.38, No. 3, 1992 403
mm to hydrolyze inulin to fructose, and centrifuge the mixture at 4#{176}C. Mix 100 ML of the supernate with 500 1zL of reagent [200 mmolfL triethanolamine . HC1 buffer, pH 7.6, containing 4 inmol of MgSO4, 1.5 mmol of NADP, 5 mmol of ATP, 2.2 kU of hexokinase (EC 2.7.1.1), and 1.1 kU of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) per liter]. Read the absorbance (A1) at 340 nm when the endpoint of the reaction has been reached (after about 15 mm) and then add 4 1tL of phosphoglucose isomerase (700 kU/L). Read the absorbance (A2) again after 15 mm. From the difference between A2 andA1 (LA) for sample and blank, calculate the inulin concentration from the molar absorptivity of the NADPH formed (6.3 x iO L mol’ cm1); subtract the value for the serum sample before the inulin injection (the endogenous fructose content). Chemical method: Incubate the sample with the glucose-removing reagent as described above. Then add 350 1.tL of 0.612 mol/L trichloroacetic acid solution and centrifuge after 15 mm. Mix 200 ML of the supernate with 500 MLof cooled anthrone reagent (5.15 mmol/L in concentrated sulfuric acid) and incubate at 37 #{176}C for 60 mm. Read the absorbance at 623 tim and calculate the concentrations by comparison with the absorbance for a 10 mg/L inulin standard. As in the enzymatic method, subtract the value for the serum sample before the inulin injection. The reaction conditions of this method (ratio of sample in reaction mixture, deproteinization) have been optimized to get high analytical sensitivity without precipitating the anthrone. Calculation of GFR All GFR values were corrected to a body surface area of 1.73 m2. A standard mTcDTPA clearance calculated by the single-compartment model analysis of the disappearance curve of mTc.DTPA was used as the comparison value for GFR (11). We injected 20 MBq of mTc. DTPA into the medial antecubital vein and measured the radioactivity in blood samples taken 60,90, and 120 mm after injection. As shown in previous studies, this technique gives values identical to those obtained in the standard inulin method involving continuous infusion (11). The single-injection inulin clearance was calculated from the plasma elimination kinetics of inulin after injection of 5 g of inulin. Timing of the clearance started with the beginning of the injection. Samples for the determination of mum concentrations were taken at seven intervals (20, 45, 90, 120, 150, 180, and 240 mm) after injection (Figure 1). GFR was calculated by applying the one-compartment model often used to approximate the inulin elimination kinetics (e.g., 6, 13). A nonlinear regression procedure was used for calculation (14): C
(PIV’)e
-
(GW’)t
(1)
where C (mg/L), P (mg), V’ (L), and G (mL/min) correspond to the inulin concentration, doses of injected 404
CLINICALCHEMISTRY,Vol.38, No.3, 1992
E a a a
90 Time
120 150 180 (mm)
240
after injection
Fig. 1. Example of inulin eliminationfrom serum after a single
injection of 5 g of inulin Bloodsampleswere drawn at the indicated times
inulin, the distribution volume of inulin in the individual, and the GFR of the individual, respectively, and t indicates the sampling time after injection. Using the measurements at the times tm and t yields the following equations: C
=
(PIV)e
-
(G’IV’)t,,
(2)
C
=
(P/V)e
-
(G’/V’)t
(3)
Dividing equation logarithm yields:
2 by equation
GIV V
=
=
(tm
G(tm
Substituting for G/V numerator of equation
-
-
3 and deriving
the
tY’ln(C/C)
(4)
t)/ln(C/C)
(5)
in the exponent 2 yields equation
and V2 in the 6:
P ln (CIC)
(6) C’t(tm
-
t)e”1t”
-
t) ID(C4,/C,,,)
we also calculated mum clearance a two-compartment model in which all
For comparison,
by applying
seven inulin concentrations
measured
were used (14).
Statistical Calculations Correlation and regression analyses between GFR determined by the Tc-DTPA method and that obtained with the single-injection inulin clearance were carried out with the nonparametric procedure of Passing and Bablok (15). Statistical differences were evaluated with Student’s t-test of paired data. Results and DiscussIon Figure 1, showing the kinetics of inulin disappearance from serum after a bolus injection of 5 g of inulin,
is a typical curve of plasma clearance. Plasma clearance is defined as the volume of plasma cleared of the respective substance per unit of time without taking into account the organs involved in the elimination of the substance. Renal clearance considers only that volume of plasma cleared by the kidney. Plasma clearance and renal clearance are identical only if the substance injected is eliminated only through the kidney. Because inulin fulfills the conditions of a classical ifitration marker and because inulin clearance has long been recognized as the “gold standard” in measuring GFR (5), measurement of the plasma clearance of mum has also been recommended as a plasma clearance method (4, 6-8, 10). This approach avoids the two principal disadvantages of the conventional mum clearance method: the continuous intravenous administration of inulin and the necessity of the exact collection of urine samples by either vesical catheterization or a previous water-loading procedure (1). However, the analytical difficulties of measuring inulin in low concentrations have limited its use (9,10,16). In addition, the results of such studies have been inadequately documented or have not been compared with standard clearance techniques (4, 7,8, 10). In this study, we used the 9’Tc-DTPA method for comparison, which correlated well with the classical inulin clearance method (11). Compared with other radioactive tracers such as 51Crlabeled ethylenediaininetetraacetic acid and [‘2511iothalamate, plasma clearance of mTcDTPA closely approximates classical renal clearance (5). Thus, we chose the Tc-DTPA clearance method as the comparison method. The mathematical analysis of the disappearance curves of inulin (Figure 1) or other filtration markers shows two exponential terms, one fast and one slow, according to an open two-compartment system (1, 17). These terms reflect two underlying processes in the elimination of inulin: the equilibration of its concentration in plasma with its concentrations in other organs and interstitial space, and its renal excretion. Numerous blood samples must be analyzed to define precisely the elimination kinetics and to determine these two terms (6). Taking into consideration all inulin concentrations measured at the seven sampling times (Figure 1), we calculated the GFR values by a nonlinear regression procedure (14). This GFR corresponded to the results of the ‘Tc-DTPA method, the 95% confidence intervals of the intercept and slope of the nonparametric regression being 1 and 0, respectively (Figure 2). However, this approach, requiring numerous samples, is not suited for clinical practice. Therefore, a one-compartment
system
is instead
recommended
(3-6),
which allows us to calculate clearances with fewer samples. We used a two-point method based on the one-compartment model to determine the optimal sampling times that would yield corresponding values between mum and DTPA clearance (see Materials and Methods).
Because
the results
obtained with this calculation
V
= a
:.i:.
LW
a a a a
jy=i.ii x + 0.54
/ 50
100
150
200
clearance (mL/min) Fig. 2. Single-injectlon inulin clearance calculated by the twoDTPA
compartment method of Inulineliminationkineticscomparedwiththe
standardclearanceof ssmTc.DTPAIn 21 patients The values for inulinclearancewere obtained by analyzing the inulineliminationkineticswithuse of all seven time poInts (14) IndIcated in Fig. 1. -, regression line according to Passing and Bablok(15);- - - -, line of Identity
model depend on the sampling times (4,13), we used all 21 two-point combinations of the seven data points to determine the optimal combination of two sampling times that would yield GFR results deviating the least from the GFR determined by the Tc-DTPA method. The optimal pair of time points, chosen from the resulting 21 linear-regression analyses according to Passing and Bablok (15), were 20 and 240 miii after injection
(Figure 3B). Figure 3 illustrates the importance of the sampling time on the resulting GFR values. For the combination selected (Figure 3B), the 95% confidence interval of the slope (0.84 to 1.09) includes 1.0 (i.e., the line of identity); similarly, the corresponding confidence interval of the intercept (-6.6 to 10.4) includes 0. Thus, at a significance level of 0.05, the data show we cannot reject that the mum clearance model is equal to the 1TcDTPA method. In addition, the percentage deviation of mum clearance from the DTPA clearance does not seem to depend on the value of the GFR (Figure 4). Using the two optimal sampling times, 20 and 240 miii after the injection of 5000 mg of inulin, we can calculate GFR (mL/min per 1.73 m2) from equation 6 as follows:
GFR =
BS- 22 730 - ln(C C=2o
-
=
2/C
eOO9Obo_o)
where BS is the factor to correct the GFR to a body surface area of 1.73 m2. After submitting this paper, we received information on two recent publications in which similar approaches of a single-injection inulin method were used (18, 19). The results described in those papers closely agree with our data. In summary, our study demonstrates that a singleinjection inulin clearance based on measuring only two blood samples provides reliable information on GFR. CLINICAL CHEMISTRY, Vol. 38, No. 3, 1992 405
a
75
I
6
:
50
E
25
. .
S
=
a a
a
e
a
#{149} .
U
.
1
-75 -‘vu, ‘nfl
clearance
I
.
a
DTPA
. .
-25 -50
=
. -.
.#{149}._
__
I i#{243}o
150
200
DTPA dearance (mIImln)
(mL/min)
Fig. 4. Percentagedeviationof the single-injection inulin clearance, calculatedby the two-point method at 20 and 240 mm, from the ‘Tc-DTPA clearance = References 1. Bianchi C, Donadio C, Tramonti G. Nomnvasive methods for the measurement of total renal function. Nephron 1981;28:53-7. 2. Van Lente F, Stilt P. Assessment of renal function by serum creatimne and creatimne clearance: glomerular filtration rate estimated by four procedures. Cliii Chem 1989;35:2326-30. 3. Apple FS, Benson P, Abraham PA, Rosano TG, Halstenson CE. Assessment of renal function by inulun clearance: comparison with creatinine clearance as determined by enzymatic methods. Clin Chem 1989;35:312-4. & Rosenbaum JL, Kramer MS, Raja EM, Manchanda R, Lazaro N. Determination of inulin and p-aminohippurate clearances without urine collection. Nephron 1973;1O:347-54.
E a
a a a
DTPA clearance
5. Levey AS. Use of glomerular filtration rate measurements to assess the progression of renal disease. Semun Nephrol
(mL/min)
1989;9:370-9.
6. Hall JE, Guyton AC, Farr BM.A single-injection method for
measuring
glomerular
filtration
rate. Am J Physiol
1977;232:
F72-6. 7. Svenningsen NW. Single-injection polyfructosan clearance in normal and asphyxiated neonates. Acta Paediatr Scand 1975;64:
87-95. GI, Jeck D, Earon J, et al. Simultaneous measurement of glomerular filtration rate and renal plasma flow using plasma disappearance curves. J Pediatr 1973;83:749-57. 9. Jung K, Klotzek S, Schuize BD. Refinements of assays for low 8. Silkalns
-2/
SW
Ly2x vO
50
100
DTPA clearance
-
-4&4 150
200
(mLjmin)
FIg. 3. Single-injectioninulin clearancecalculatedby the two-point methodof inulineliminationkinetics,comparedwith data for semTc DTPA clearancein 21 patients GFR values were calculated on the basis of the two-pointcombinationsof 20 and4smln(A), 20and24Omin(, and l5Oand24Omin(C).-, regression IlneaccordlngtoPasslngandBablok(l5);----, Ilneofidentity
we included only subjects with GFR values >35 mL/min in our study, the reliability of this approach is proved, at least for this value for GFR. The recommended procedure is simple and expeditious and is suitable for routine use. It could replace unreliable Because
measurements
of endogenous to
20) and is an alternative
creatinine
clearance
isotope techniques.
406 CLINICALCHEMISTRY,Vol.38, No. 3, 1992
(2,3,
concentrations of inulin in serum. Nephron 1990;54:360-1. 10. Yataidis H. Simplified determination of plasma inulin in testing kidney function. Clin Chem 1976;22:1239. 11. Schulze BD, Precht K, Sydow K, Jung K. Diagnostische Moglichkeiten zur approximativen Bestimmung der glomerulfiren Filtrationarate. Z Klin Med 1991;45:1717-9. 12. Lewis R, Kerr N, Van Buren C, et al. Comparative evaluation of urographic contrast media, inulin, and Tc-DTPA clearance methods for determination of glomerular filtration rate in clinical transplantation. Transplantation 1989;48:790-6. 13. Tepe PG, Tauxe WN, Bagchi A, Rezende P. Krishnaiah PR. Comparison of measurement of glomerular filtration rate by single sample, plasma disappearance slope/intercept and other methods. Eur J Nucl Med 1987;13:28-31. 14. Holzh#{252}tter HG,Colosimo A. SIMFfl’ a microcomputer softwaretool kit for modelistic studies in biochemistry. Cabioe 1990;6:23-8. 15. Passing H, Bablok W. A new biometrical procedure for testing the equality of measurement from two analytical methods. J Cliii Chem Clin Biochem 1988;21:709-20. 16. Gibb DM, Dalton NB, Barratt MT. Measurement of glomerular filtration rate in children with insulin-dependent diabetes mellitus. Clin Chim Acta 1989;182:131-40. 17. Sapirstein LA, Vidt DG, Mandel MJ, Hanusek G. Volumes of distribution and clearances of intravenously injected creatinine in the dog. Am J Physiol 1955;181:330-6. 18. Gretz N, Ecker-Tschinner KH, KUhnle HF, et al. Practicability
of the
inulin plasma single-shot clearance. Contrib Nephrol 1990;81:220-8. 19. Prescott LF, Freestone S, McAualane JAN. Reassessment of the single intravenous injection method with inulin for measure-
ment of the
glomerular filtration rate in man. Clin Sci 1991;80:167-76. 20. Levey AS, Perrone RD, Madias NE. Serum creatinine and renal function. Annu Rev Med 1988;39:465-90.
CLIN. CHEM. 38/3, 407-411(1992)
Total Urinary Hydroxyproline Determined with Rapid and Simple High-Performance Liquid Chromatography R. Paroni, E. De Vecchi, I. Fermo,
C. Arcellom, L. Diomede,’ F. Magni, and P. A.
A precolumn denvatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N.diethyl-2,4-dinitro-5-fluoroaniline (FONDEA) at 100 #{176}C for 20 mm. The DNDEA-hydroxyproline adduct
was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitnle (80/20, by vol), and was detected at 360 nm. A single run took 18 mm with a hydroxyproline retention time of 7.3 mm. The assay
showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y vs ion-exchange chromatography with postcolumn ninhydrin reaction (,) correlated well: y = 0.98x + 1.02 (r = 0.985, Si,, = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon#{174}, x) gave slightlyhigher values than the HPLC method (: y = 0.83x + 2.21 (r = 0.937, S,, = 4.6). AddItIonal Keyphrases: bone protein In humans,
chromatography,
reversed-phase
amino acids most of the body collagen
is in bone, which
represents the major reservoir of this protein (1). About 14% of the total amino acids content of collagen is 4-hydroxyproline (2), derived from hydroxylation of proline residues after protein is synthesized (3). Because the hydroxyproline released from collagen degradation is not reused, but only catabolized and excreted, the amounts of free hydroxyproline and hydroxyprolinecontaining peptides in urine are strictly related to Istituto Scientifico
H. San
Raifaele,
via Olgettina
60, Milano,
Italy. ‘Istituto di Ricerche Farinacologiche Mario Negri, via Eritrea 62, Milano, Italy. Received July 1, 1991; accepted January 7, 1992.
collagen metabolism (4, 5) and to bone-related diseases (2,6). Quantification of urinary 4-hydroxyproline allows diagnosis and monitoring of a variety of diseases, such as osteoporosis, Paget disease (2), bone secondary cancers (7), and hereditary disorders (8). Early colorimetric methods for urinary hydroxyproline determination involved oxidation of hydroxyprolme followed by colorimetric reaction with Ehrlich’s reagent (9). Because many urinary components can interfere
with these assays, different modifications of the original method were proposed (10-13), including a preliminary purification of urine hydrolysates before analysis (14) with an ion-exchange sulfonic column. Recently several high-performance liquid.chromatographic (HPLC) methods have been developed. These assays combine the good resolution specificity
power of HPLC with the sensitivity and of various derivatizing agents, e.g., dabsyl chloride (15-18), phenylisothiocyanate (19-21), dansyl chloride (22), 4-chloro-7-nitrobenzofurazan (23-25), and 9-fluorenylmethyl chloroformate (26-28). Most of these derivatization methods present problems with quantifi-
cation and require an initial cleanup step with o-phthaldialdehyde, which removes interfering peaks by selectively reacting with primary amino acids (26,28). Our purpose was to obtain a sensitive, specific, and rapid HPLC method for determination of total urinary
4-hydroxyproline.
We recently
suggested
the use of
N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) as a precolumn derivatizing agent that allows determination of primary and secondary amino acids (29). In addition we tested the reliability of this procedure for quantifying amino acids in serum (30). Here we present a modification of the previous HPLC method, designed to quantify hydroxyproline in hydrolyzed urine samples. In this method, the hydroxyproline peak is not affected by interfering compounds, so the preliminary purification step with o-phthaldialdehyde is avoided.
Materials and Methods Chemicals. 4-Hydroxy-L-proline and L-cysteic acid were from Sigma Chemical Co. (St. Louis, MO). FDNDEA and triethylamine were purchased from Fluka (Buchs, Switzerland). Sodium hydrogen carbonate, acetic acid, CLINICAL CHEMISTRY, Vol.38, No. 3, 1992 401
