Kidney International, Vol. 46 (1994), PP.

252259

TECHNICAL NOTE

Glomerular filtration rate measurement by "single-shot"

injection of inulin
KLAAS W. FLORIJN, Jos N.M. BARENDREGT, EEF G.W.M. LENTJES, WIM VAN DAM,
WIGuN0 PRODJOSUDJADI, JAN L.C.M. VAN SAASE, LEENDERT A. VAN Es,
and PETER C. CHANG

Department of Nephrology, Department of Clinical Epidemiology, and Department of Clinical Chemistry, University Hospital Leiden,
The Netherlands

Glomerular ifitration rate measurement by "single-shot" injection of a major practical problem, limiting the reproducibility of the
Inulin. In 14 ADPKD patients the total body clearance and the urinary constant infusion and other urinary clearance methods. Inter-
clearance of inulin using the constant infusion method were compared
with the "single-shot" technique. Triplicate measurements of both estingly, limited data are available about the reproducibility of
clearances by each infusion method were obtained in 12 out of 14 the "gold standard" and alternative techniques in the same
patients. A high correlation was found between the total body clearance patient groups [17]. For measuring the progression of renal
and the urinary clearance for both the constant infusion method (r = failure the reproducibility of the GFR measurement within an
0.96) and the single injection technique (r = 0.96). The coefficient of individual is of key importance, as this will strongly influence
variation for the total body clearance of inulin was significantly lower
for the constant infusion method and the single injection technique the needed sample size of intervention studies [18].
(7.8% and 7.1%) than for the urinary clearance of inulin (11.3% vs. The primary aim of the present study was to define and
9.7%, P < 0.05). A constant overestimation of the urinary clearance by compare the agreement and reproducibility of the single intra-
the total body clearance was observed with both methods (constant venous injection of inulin with the method using a constant
infusion method 8.3 ml min' . 1.73 m2 and single injection tech- infusion of inulin.
nique 13.4 ml min 1.73 m2). No concentration-dependent clear-
ance was present. Determination of plasma inulin, especially at low
levels, showed substantial interference by glucose. We conclude that, Methods
taking into account a constant overestimate of urinary clearance by the The study was approved by the Medical Ethics Committee of
total body clearance of inulin, the single injection total body clearance
possesses the best reproducibility and shows a good agreement with the University Hospital Leiden.
the conventional urinary clearance, which can be calculated by: GFR =
TBCLss 13.1 ml min' . 1.73 m2 (in the range of 28 to 124 Subjects
ml min' . 1.73 m2). Fourteen patients with autosomal-dominant polycystic kid-
ney disease (ADPKD), defined by positive family history and
characteristic abnormalities on sonography, and three patients
with non-insulin dependent diabetes mellitus (NIDDM) were
It is well recognized that serum creatinine and even creati-
studied (Table 1). All gave informed consent prior to the study.
nine clearance are of limited value for assessing the level of
None of the participants took drugs (other than oral contracep-
renal function and the rate of progression of renal disease [181.
tives in the case of females) on the study days. Antihyperten-
Thus, an increasing number of investigators use the glomerular
sive treatment was discontinued for at least one week.
rate (GFR) as a more accurate measuare to evaluate the efficacy
of interventions aimed at preventing or retarding the progres- Study design
sion of renal failure [9131.
GFR is measured using a variety of methods and filtration In 12 patients with ADPKD the clearance of inulin was
markers. Urinary clearance of inulin, using a constant infusion, measured three times with the constant infusion method and
has long been recognized as the "gold standard" [2]. Several three times with a single intravenous injection technique on
difficulties with this method have led to the development of separate occasions over the course of six weeks. Every other
alternative methods [5, 1416]. First, glucose interferes with the
week one constant infusion and one single injection technique
measurement of inulin. Second, adequate collection of urine is of inulin, in random order, was performed and these values
were used for comparison. To study the clearance of inulin at
high plasma levels, in three of these patients (patients 1, 2, and
3; Table 1) the constant infusion was repeated 12, 12, and 7
Received for publication November 20, 1992 months after the initial measurements, with a five- to sixfold
and in revised form February 16, 1994 loading and maintenance dose of inulin. The remaining two
Accepted for publication February 17, 1994 ADPKD patients had one single intravenous injection and one
1994 by the International Society of Nephrology constant infusion of inulin in random order. In these patients,

252
 Florijn et a!: Single-shot of inulin 253

Table 1. Clinical characteristics of patients

Age Body weight BSA Serum creatinine Creatinine clearance

Patient no. Diagnosis years Sex kg m2 .unol/liter ml/min/1.73 m2
1 ADPKD 43.8 f 62.0 1.7 197 36
2 ADPKD 33.0 m 73.0 1.9 181 39
3 ADPKD 34.1 f 81.5 1.9 84 89
4 ADPKD 40.0 m 80.0 2.1 94 104
5 ADPKD 47.3 f 72.0 1.7 133 40
6 ADPKD 62.2 f 75.0 1.8 127 47
7 ADPKD 57.7 f 76.5 1.9 70 86
8 ADPKD 43.7 f 64.0 1.8 69 99
9 ADPKD 67.4 m 84.0 2.1 146 42
10 ADPKD 26.5 f 67.5 1.8 82 97
11 ADPKD 36.6 m 80.0 2.0 119 70
12 ADPKD 29.4 m 81.0 2.0 142 89
13 ADPKD 38.4 f 52.0 1.6 80 56
14 ADPKD 68.8 m 80.0 1.9 210 44
15 NIDDM 59.6 in 75.0 1.9 244 25
16 NIDDM 57.8 f 121.0 2.4 109 46
17 NIDDM 67.5 m 78.5 2.0 147 50
Abbreviations are: ADPKD, autosomal-dominant polycystic kidney disease; NIDDM, non-insulin dependent diabetes mellitus; BSA, body
surface area.

inulin was assayed with the specific inulinase method [19]. To Constant infusion
study the influence of plasma glucose on the determination of
inulin by the non-specific anthrone method, three patients with After a loading dose, as described above, a constant infusion
non-insulin dependent diabetes mellitus were administered a was started at a rate adjusted to maintain plasma concentrations
single intravenous injection of inulin and infused 25 grams of inulin at 300 mg/liter, calculated as:
glucose during the elimination phase, from t = 180 until t = 200 Infusion rate (mg/mm) 0.3 * Cockcroft clearance (mllmin).
minutes.
After equilibration for 70 minutes, four clearance studies were
Inulin clearance measurements performed, each lasting 30 minutes. The average of these four
All patients fasted on each study day from 0.00 hours until the clearances was taken for further analysis.
end of the inulin clearance measurement. After awakening, they
drank 500 ml of water and reported to the unit at 08.30 hours. Laboratory methods
Twenty-four-hour urine collection started after voiding. To
enhance diuresis in the ADPKD patients, 1 liter of glucose 5% Plasma and urine were either stored at 20C and assayed for
was infused in 30 minutes, prior to the start of the inulin creatinine and inulin within 72 hours, or stored at 70C until
infusion. To ensure ample diuresis in diabetics, these patients analysis. Serum and urine concentrations of creatinine were
drank 1000 ml water prior to the start of the inulin infusion. determined by a standard autoanalyzer technique. Inulin in
Each patient was studied in the same position, either supine or plasma and urine samples of all patients, except the NIDDM
sitting during each study day, except when voiding. The inulin patients, were measured using a specific enzymatic method. In
loading dose was determined using the calculated lean body this assay, inulinase hydrolyzes inulin to its fructose mono-
mass [20], as: mers, followed by enzymatic conversion of fructose to glucose-
6-phosphate which can be determined with the aid of glucose-
Loading dose (mg) = 400 * Inulin space (L) and
6-P-dehydrogenase [19].
Inulin space (L) = 0.175 * Lean body mass (kg) To study the interference of glucose on plasma inulin levels
measured with the anthrone method, the plasma and urine
to achieve comparable plasma levels in all patients. samples of the patients with NIDDM were determined in this
non-specific colorimetric assay, with and without correction for
Single intravenous injection serum glucose. This procedure includes deproteinization, after
Inulin (Inutest', Laevosan GmbH, Linz, Germany) was which inulin and glucose are measured in the same urine or
infused at a constant rate over five minutes by a precise plasma sample by two parallel autoanalyzer units, one measur-
volumetric infusion pump (Sage Instruments, Massachusets, ing glucose with the standard method from Technicon using
USA) after which the infusion line was flushed with saline. At 0, hexokinase, and one measuring inulin after acid hydrolysis to
5, 7, 10, 20, 40, 60, 90, 120, 140, 160, 180, 210 and 240 minutes, fructose using anthrone [21]. Inulin-like optical density of
6 ml of blood were collected from an antecubital vein, in the glucose standards were used to construct a glucose correction
arm opposite in which inulin was infused. The patients were curve. Inulin concentrations of infused solutions were also
asked to void before and 60, 120, 180 and 240 minutes after the determined. Blank values of urine and plasma were used for
start of the inulin infusion. subtraction.
254 Florijn et al: Single-shot of inulin

////
150 150
/
0
125
.
// 0
125
-c
ci,

C . /
./
U)
ci)
.
/
100 0)
C
100

U)
75
;S a) . 75

a)
E 50 E 50
-j -J
C)
/S r=0.96 0 r=0.96

/ /
I.- I-
25 25

0 0
0 25 50 75 100 125 150 0 25 50 75 100 125 150
UCL measured with C-infusion UCL measured with single shot
ml/min/1.73 m2 mI/mm/i. 73 m2
Fig. 1. The total body clearances (TBCL) of all individuals versus the Fig. 2. The total body clearances (TBCL) of all individuals versus the
urinary clearances (UCL) during the constant infusion of inulin. urinary clearances (UCL) during the single injection technique of
Included in the graph is the line of identity. inulin. Included in the graph is the line of identity.

Pharmacokinetic analysis Plasma samples were obtained at the beginning and end of
Urinary clearances of inulin were obtained by dividing the every urinary collection period. All clearances were normalized
urinary amount of inulin excreted over time by the time- to a standard body surface area (BSA) of 1.73 m2, using the
averaged plasma concentration calculated with the trapezoidal DuBois-DuBois formula for BSA [231.
rule. Total body clearance of inulin during the single intrave-
nous injection was calculated by dividing the infusion dose with Statistics
the area under the curve, obtained by curve fitting. The plasma
decay curves were fitted to a two-compartment pharmacoki- All values are expressed as mean SD, except when speci-
netic model with zero-order administration of the dose over five fied otherwise. Correlation coefficients were used to describe
minutes, using the SIPHAR program (Simed, Crteil, Cedex, the strength of the relation between the single intravenous
France). This method is based on two simultaneous exponential injection technique and the constant infusion method of inulin.
processes with constant rate input, defined as: Agreement of the total body clearance of inulin using the
intravenous injection technique and the urinary clearance of
B(l eIta * T)ebeta(t T)

A(l e_a1) * T)ealPha(t T)
inulin using the constant infusion method were evaluated by
= + limits of agreement and graphically represented as described by
alpha * T beta * T
Bland and Altman [24]. These limits of agreement represent the
where C the plasma inulin concentration at time t, T = the range around the mean difference (D) between both methods in
duration of infusion, being five minutes, with A, alpha, B, and which 95% of the values will be found. They are defined as D
beta being hybrid constants [22]. They were first obtained by 1.96 * SD when the differences between both methods approx-
"peeling" and subsequently refined by iterative non-linear imate a Gaussian distribution. Reproducibility of both methods
regression analysis using the Powell minimization algorithm was tested by comparison of the coefficients of variation using
with a weighting factor of l/(calculated concentration)2 Ini- the Kruskal-Wallis non-parametric test.
tially, all plasma values were used for curve fitting, followed by
the same procedure with seven samples, including the blank. In Results
this analysis, two values were taken to describe the distribution
phase and four samples to describe the elimination phase in the Total body versus urinary clearance of inulin
time period of 90 to 180 minutes. Thus, the total amount of time
required for the single injection technique equalled the time Total body clearance of inulin overestimated urinary clear-
period used during the constant infusion clearance. The total ance at all levels of GFR, both during constant infusion (Fig. 1
body clearance of inulin during the constant infusion was and Table 2) and single intravenous injection (Fig. 2 and Table
calculated from the concentration of the infusate times infusion 2). The degree of overestimation was not related to the level of
rate, divided by the time-averaged plasma concentrations. GFR (Figs. 1 to 3). The mean difference of single injection total
 Florijn et al: Single-shot of inulin 255

Table 2. Comparison of inulin clearances

Cv CV CV Cv
Patient no. UCLci % TBCLci % UCLss TBCLss %
1 27.9 17.2 27.3 6.9 27.1 17.9 41.8 7.4
2 36.3 8.7 46.1 10.0 34.8 0.6 49.2 5.3
3 93.1 16.2 113.6 4.7 98.6 12.6 107.8 8.8
4 95.5 11.3 107.2 10.2 88.6 8.8 120.2 4.8
5 38.1 11.8 51.0 5.6 39.8 10.1 48.7 10.7
6 44.2 8.8 43.3 9.4 52.0 5.4 53.0 6.0
7 91.4 7.9 92.2 11.3 80.4 10.4 86.7 9.2
8 123.4 5.1 136.9 6.9 111.4 9.4 129.5 10.8
9 51.7 13.9 53.7 7.7 56.0 9.0 66.7 0.7
10 107.9 10.1 128.9 4.2 110.9 19.9 127.3 10.7
11 59.6 16.9 65.2 10.7 62.3 3.4 73.3 6.1
12 74.9 8.2 82.3 5.5 77.5 9.0 97.0 5.1
13 71.8 71.2 72.2 86.2
14 39.3 41.1 45.2 60.7
Mean 11.3 7.8 9.7 7.1
sn 3.9 2.5 5.4 3.1
All clearances are expressed as ml/minll .73 m2. Abbreviations are: CV, variation coefficient; UCLci, urinary inulin clearance during constant
infusion; TBCLci, total body clearance during constant infusion; UCLss, urinary inulin clearance during single-shot; TBCLss, total body clearance
during single-shot.

body clearance minus urinary clearance during constant infu- intravenous injection and 7.8% during constant infusion, com-
sion, was 13.1 ml min' 1.73 m2 (Fig. 3). Limits of agree- pared with 9.7% and 11.3% for the respective urinary clear-
ment were 7.7 and 33.9 ml min1 1.73 m2. The overesti- ances (Table 2). The variation coefficient of the total body
mation by the total body clearance of urinary clearance was on clearances and the urinary clearances of both methods differed
average 8.3 ml mint 1.73 m2 when the constant infusion significantly (P < 0.05). The variation coefficient of the total
method was used, and 13.4 ml min1 1.73 m2 for the single body clearance was not significantly different with 6.6 3.4%
injection technique. Correlation between total body and urinary when total body clearance during the single injection technique
clearance of inulin was high, r = 0.96 during the constant was calculated with seven instead of 14 plasma samples.
infusion, and r = 0.96 for the single injection technique. Inulin
recovery from the collected 24 hour urine was 88.2 17.4% in Effect of five- to sixfold higher plasma inulin levels on urinary
case of the single-injection technique (Fig. 4). mu/in clearance
To evaluate linearity of inulin kinetics, in three of the ADPKD
Comparison of the single injection and constant infusion patients we repeated a constant infusion study with five- to sixfold
methods higher loading and maintenance doses of inulin. In all three
Urinary clearances of inulin measured with both methods did patients stable plasma concentrations were reached, with levels of
not differ significantly; the same was observed for total body 1337 to 1965 mg/liter. Urinary inulin clearance in these patients
clearances (Figs. 5 and 6). Mean creatinine clearances during (Patients 1 to 3; Table I), determined 12, 12 and 7 months after the
both methods were similar and not significantly different. initial study period, and calculated as the mean of four individual
Because of the substantial number of samples (N = 14) used clearance periods, were similar to previous values (27.2, 35.3 and
in the single injection method, we studied the effect of reducing 90.9 m1 1pj11. 1.73 m2 respectively, which is 0.7, 1.0, and 2.2
the number of plasma samples to a similar number of plasma ml miir1 1.73 m2, respectively, lower than in the initial
samples used in the constant infusion method on the main study).
outcome parameter, that is, total body clearance of inulin.
Describing the distribution phase by two samples at t = 10 and Effect of glucose on plasma inulin values
20 minutes, and the elimination phase by four plasma samples at To evaluate the influence of plasma glucose on plasma inulin,
t = 90, 120, 160 and 180 minutes, an excellent agreement of total determined by the anthrone method, and the clearance values,
body clearance of inulin calculated with the total body clear- the effect of an intravenous infusion of 50 ml glucose 50% over
ance determined with all plasma samples was found (r = 0.996, 20 minutes was examined during the elimination phase of inulin
Fig. 7). An additional advantage of this reduced number of (Fig. 8) in three NIDDM patients [mean 24-hr creatinine clear-
samples is that it takes less time for performing a single ance 40.5 8.0 ml min' 1.73 m2 (mean sEM)]. This
injection method (3 hr instead of 4 hr), equalling that of a resulted in an increase of plasma glucose from 6.8 0.6
constant infusion procedure. mmollliter to 13.2 0.2 mmollliter (mean SEM). The concom-
itant values of plasma inulin measured with the anthrone
Reproducibility of the single injection and constant infusion method, uncorrected for glucose, rose from 121.7 23.0
methods mg/liter to 180.0 26.7 mg/liter (mean SEM). The interference
The results of triplicate estimations of the inulin clearance by by glucose on urinary inulin clearance, especially in this time
both methods are shown in Table 2. Mean coefficient of period (that is, 180 to 240 mm) was substantial. With correction
variation for total body clearance was 7.1% during single for glucose, mean urinary inulin clearance was 43.6 3.1
256 Florfjn et a!: Single-shot of inulin

50 150
(I)
U)
-J 40
0
30
.. 125
C
20 ... . 0
-c
U)

a)
a)
0C
10 . S.
a)
C 100
0 SC')
a)
a) 10

20 I I
.S I I
a) .
75

0 25 50 75 100 125 150

E 50
Mean of TBCLss and UCLc1, mI/mm/i. 73 m2 -J
0
Fig. 3. Difference against mean for total body clearance during the I
single injection (TBCLss) and urinary clearance during the constant 25
infusion method (UCLci). Dashed lines represent the limits of agree-

t.
ment around the averaged difference.
120 0

S . 0
S 25 50 75 100 125 150

100
. TBCL measured with C-infusion
0
-C S mI/mm/i. 73 m2
S S Fig. S. Comparison of total body clearances (TBCL) obtained with the
c'j
80 . S single intravenous injection and the constant infusion method. Also

0EU)
C
2.2
60 S S
.
S depicted is the line of identity.

Wa) level of GFR, as shown in Figures 1 and 2. An overestimation

>C
o -C has also been observed for other glomerular filtration markers
a)
40 like mTcDTPA and 51Cr-EDTA [15]. In the same study,
C inulin urinary clearance was 43.6 mllmin (range 11.0 to 76,1
C 20 mllmin) in 20 patients and plasma clearance 53.8 mllmin (range
25.1 to 83.5 mI/mm) [15]. A similar degree of overestimation by
total body clearance over a broad range of GFR, leading to a
0 close correspondence of total body inulin clearance with the
0 25 50 75 100 125 150 classical endogenous creatinine clearance, was demonstrated
by Silkalns et al [25]. The overestimation of GFR by the total
UCL measured with single shot body clearance has also been confirmed in subjects with a
mI/mm/I. 73 m normal renal function, their total body clearance being 144 15
Fig. 4. Inulin recovery from 24-hour urine after a single intravenous ml/min and urinary clearance 125 20 ml/min [26]. The same
injection in relation to the observed urinary clearance (UCL).
observation was recently extensively confirmed over a broad
range of GFR by Hellerstein et al [27], who suggested that this
may be the result of incomplete equilibration of inulin in the
ml min 1.73 m2, and 28.9 9.4 ml minj 1.73 m2 (mean extracellular volume, requiring 12 to 15 hours or possibly even
sEM) without correction for glucose. Plasma inulin values were longer. Thus, penetration of inulin into the less permeable
practically identical with the extrapolated values (127.3 20.1 components of the interstitial matrix of the extracellular fluid
mg/liter vs. 121.7 18.8 mg/liter), when corrected for glucose. compartment will be highest with increasing time. The resultant
overestimation of the urinary clearance by the total body
Discussion clearance will be less with the constant infusion technique than
The main findings of this study are that: (1.) there is a with the single injection method, as measurement of GFR by
constant overestimation of urinary clearance by the total body the constant infusion method starts after an equilibration period
clearance derived from either the constant infusion method or of 70 minutes. This was confirmed by our data, the difference
the single injection technique; (2.) there is a very close corre- between total body clearance and urinary clearance being
lation of the single injection total body clearance with the largest for the single injection technique (13.4 vs. 8.3
urinary inulin clearance using the constant infusion method ml min 1.73 m2, P < 0.05). The additional observation that
("gold standard"); and (3.) the total body clearance of inulin inulin recovery in the 24 hour urine was complete fits well with
has a much better reproducibility than the urinary clearance of the explanation of incomplete equilibration of inulin in the
inulin. extracellular volume, as extrarenal inulin clearance would
The degree of overestimation seems to be unrelated to the result in incomplete inulin recovery.
 Flor,jn et a!: Single-shot of inulin 257

150 175

125
. 0
U,
150

Co

100 1.
. 0
125

/. H 100

(
'I)

75 . 75
E
E
50
/
/ -J
QN.
50
,0.996
-J ,0.95 I-
0 25
25 0
0 25 50 75 100 125 150 175
0
0 25 50 75 100 125 150 TBCL measured with single shot,
all samples, mI/mm/i. 73 in2
UCL measured with C-infusion Fig. 7. Comparison of total body clearances (TBCL) obtained with the
mI/mm/i. 73 m2 single intravenous injection method, calculated with all samples (x-
Fig. 6. Comparison of urinary clearances (UCL) obtained with the axis) or seven plasma samples (y-axis). Also depicted is the line of
single intravenous injection and the constant infusion method. Also identity.
depicted is the line of identity.

In studying the single intravenous injection technique, uri- ances will be found, in particular during the elimination phase.
nary inulin clearance during the first hour, including peak The studies [3235] cited by Prescott et al, in favor of the
plasma inulin levels, was similar to urinary clearance during concentration-dependent urinary clearance of inulin used as-
subsequent hours with low plasma inulin levels in the terminal says [3638] in which interference of glucose is difficult to
elimination phase. These results are in contrast with data exclude. We suggest that the presumed concentration-depen-
presented by Prescott et al, who reported that the elimination of dent urinary clearance of inulin in these studies is most likely
inulin is highly concentration-dependent in both healthy volun- based on interference by glucose in the anthrone method. This
teers and patients with renal failure [28, 291. They found that notion is also supported by Jung, Klotzek and Schulze [39],
urinary inulin clearance decreased progressively and signifi- who removed glucose prior to the colorimetric measurement in
cantly as plasma concentrations fell over time after a single order to improve reliability of low plasma inulin levels. They
injection of inulin. In healthy volunteers this was already observed close correspondence of inulin levels measured by
apparent after two hours, in patients with renal failure approx- both the colorimetric method and a specific enzymatic inulin
imately after three hours, with plasma inulin levels in the range assay even with low plasma levels, which we could confirm
of 100 mg/liter in healthy volunteers and somewhat higher in (Lentjes et al, unpublished observations), Finally, others [40]
patients with chronic renal failure. In a further extension of repeating constant infusion urinary inulin clearance with careful
their study, using a constant infusion of inulin with plasma urine collection in stable patients, using plasma inulin levels
inulin levels between 35.2 6.9 mg/liter and 187 29.3 mg/liter ranging from 147 mg/liter to 232 mg/liter and during the second
(means SD), urinary inulin clearances were again found to be study ranging from 259 mg/liter to 491 mg/liter, also found no
lower [29]. These findings could not be confirmed in our study. significant difference in urinary inulin clearance. This confirms
A possible explanation for these discrepant findings could be our observation that urinary inulin clearance is not influenced
the difference in assaying methodology for inulin. The spectro- by the level of plasma inulin levels.
photometric method of measuring inulin used by Prescott et al To study renal handling of inulin during high plasma levels, in
is complicated by interference with carbohydrates, especially three ADPKD patients urinary clearance of inulin using plasma
glucose [30]. The amount of interference by endogenous sub- levels during constant infusion up to 2000 mg/liter were re-
stances is in the range of 5 to 15 mg/liter [31]. In our three peated. Taking into account the time interval of 12, 12, and 7
patients with non-insulin dependent diabetes mellitus, infusion months between both studies in these patients, GFR values
of glucose significantly increased inulin levels (Fig. 8). This were completely comparable with the initial measurements at
strengthens the observation that glucose in physiological con- much lower plasma inulin levels. This is in accordance with the
centrations interferes linearly with the determination of inulin unchanging values of urinary clearance during the single intra-
by the anthrone method [22]. The interference of glucose was venous injection, irrespective of high peak plasma levels or low
relatively largest when plasma inulin levels were 100 mg/liter or plasma levels during the terminal elimination phase. Using
less. As a consequence, erroneously low urinary inulin clear- higher inulin loading doses, toxicity or alteration of renal
258 Florzjn et al: Single-shot of inulin

900

800
700
600
500

I 400
300
200
-W..;
100
0.
20
15
10 Fig. 8. Effect of changes in plasma glucose
0 s-I--. I---I--I------i-- ---I-------,.. on plasma inulin levels measured with the
8 5 anthrone method in three non-insulin
Glucose i.v. dependent diabetes mellitus patients. To
0 study the influence of changing plasma
0 50 100 150 200 250 glucose levels on plasma inulin, 25 grams of
glucose was given intravenously from 180 till
Time, minutes 200 minutes after infusing inulin.

function by higher inulin concentrations were not observed, coefficient of variation of the total body clearance, this implies
even using doses as large as 160 grams [411. Obviously, at that replacing the conventional urinary clearance with the total
higher plasma inulin levels during the elimination phase less body clearance of the single intravenous injection technique
methodological assaying errors associated with low plasma will result in an improvement of the ability to detect differences
inulin levels will be found. in decline of renal function. For intervention studies, this may
From a clinical point of view it is most useful to know how permit a considerable decrease in sample size without a loss of
much a measurement with one method is likely to differ from power. However, when the exact level of GFR is required,
that obtained with another. This is expressed by the limits of conventional urinary inulin clearance may be the best option,
agreement [24]. A recent study [42] reviewing agreement limits although it can also be calculated by GFR = TBCLss 13.1
between different glomerular filtration markers found limits of ml ' min'' 1.73 m2 in the range of28 to 124 ml ' min'' 1.73
agreement between studies ranging from 5 to 25 m2.
ml' min'' 1.73 m2 and 34 to 36 ml . min ' 1.73 m2. In The reproducibility of the constant infusion of inulin and the
comparing total body clearance of the single intravenous injec-
tion technique with the conventional urinary clearance mea- single intravenous injection was good. The coefficients of
sured with constant infusion of inulin, limits of agreement of variation of total body clearances were significantly smaller
7.7 to 33.9 ml ' min 1.73 m2 were found (Fig. 3). These than the coefficients of variation for urinary clearances, despite
limits of agreement reflect both variability of the total body rigorous hydration and careful urinary collections (Table 2).
clearance of the single intravenous injection technique and of Halving the number of plasma samples during the single intra-
the urinary clearance measured with constant infusion of inulin. venous injection method had no detrimental effect on reproduc-
This means that if one method has poor repeatability, the ibility (CV of all samples 7.1% vs. 6.6% with the reduced
agreement between the two methods is bound to be poor, too. number of samples), while agreement was excellent (r = 0.996,
Thus, "when the old method is the variable one, even a new Fig. 7).
method which is perfect will not agree with it" [24]. Further- In conclusion, the total body clearance of inulin using the
more, in our experiments, day to day variability and intra- single intravenous injection method has a good reproducibility
individual variability of GFR also contribute to the variability of over a wide range of GFR, and up to 2000 mg/liter is indepen-
both methods. The difference between the total body clearance dent of plasma inulin concentrations. We observed a constant
of inulin using the single injection technique and the urinary overestimation of urinary clearance by the total body clearance
clearance obtained with the conventional constant infusion independent of the level of GFR, permitting a simple correction
method was on average 13.1 ml * min1 - 1.73 m2, and was not to estimate the urinary clearance of inulin. Careful attention is
related to the level of GFR. Taken together with the small required with regard to the interfering effect of plasma glucose
 Florjjn et a!: Single-shot of inulin
on the determination of plasma inulin levels when the anthrone
method for determination of inulin is used.
Acknowledgments
This study was supported by a grant from "Praeventiefonds Neder-
land". We thank Mrs. R. Dijkstra-Oppenhuizen who assisted in the
performance of the studies described, and Mrs. Y. Muizert for help in
performing the inulin measurements.
Reprint requests to K. W. Florijn, M.D., Department of Nephrology,
University Hospital Leiden, Building 1, C3-P, P.O. Box 9600, 2300 RC
Leiden, The Netherlands.
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