• Open Access

A Single-Blood-Sample Method Using Inulin for Estimating Feline Glomerular Filtration Rate


Corresponding author: Kazuhisa Furuhama, Department of Veterinary Basic Medicine, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; e-mail: furuhama@iwate-u.ac.jp.



Application of a multisample method using inulin to estimate glomerular filtration rate (GFR) in cats is cumbersome.


To establish a simplified procedure to estimate GFR in cats, a single-blood-sample method using inulin was compared with a conventional 3-sample method.


Nine cats including 6 clinically healthy cats and 3 cats with spontaneous chronic kidney disease.


Retrospective study. Inulin was administered as an intravenous bolus at 50 mg/kg to cats, and blood was collected at 60, 90, and 120 minutes later for the 3-sample method. Serum inulin concentrations were colorimetrically determined by an autoanalyzer method. The GFR in the single-blood-sample method was calculated from the dose injected, serum concentration, sampling time, and estimated volume of distribution on the basis of the data of the 3-sample method.


An excellent correlation was observed (r = 0.99, P = .0001) between GFR values estimated by the single-blood-sample and 3-sample methods.

Conclusions and Clinical Importance

The single-blood-sample method using inulin provides a practicable and ethical alternative for estimating glomerular filtration rate in cats.


area under the serum inulin concentration versus time curve


body surface area

CKD cats

cats with spontaneous chronic kidney disease


volume of distribution


glomerular filtration rate


blood urea nitrogen

Urinary inulin clearance for estimating glomerular filtration rate (GFR) provides the most accepted measurement of renal function.[1] However, application of the standard inulin method to feline medicine is cumbersome and difficult. For example, this procedure relies on accurately timed repeated blood and urine samplings and usually involves bladder catheterization to achieve accurate urine collection. The nonionic monomeric X-ray contrast medium iohexol has been used extensively for GFR assessment in cats,[2, 3] but using multiple (n ≥ 3) blood sampling strategies. Moreover, it has been reported that there is concern regarding the deteriorating potential of iohexol on impaired kidney function in humans.[4, 5] Use of radiolabeled compounds required specific equipment and considerations.

The concentration of a tracer in a single plasma sample taken a few hours after injection was previously reported to be well correlated with renal clearance in humans.[6] On the basis of this information, Jacobsson[7] devised a formula derived from a simple 1-compartment model combined with the volume of distribution (Vd) and optimum time for taking plasma using a radio-labeled tracer and accurately determined the GFR. Because the Vd is dependent on the elimination kinetics of each tracer and animal size, one must obtain it in the respective species.[8] The objective of the present study was to establish a simplified procedure to estimate GFR in cats based on Jacobsson's formula and using a single serum inulin concentration.

Materials and Methods

Study Design

The Jacobsson's formula for humans has not been validated for cats. To apply this formula[7] to cats, we first measured the GFR by a conventional multisample (3-, 4-, or 7-sample) method using inulin in both healthy cats and cats with spontaneous chronic kidney disease (CKD cats). Next, by substituting GFR values and serum inulin concentrations at 1 sample point into Jacobsson's formula,[7] we sought the estimated Vd in individuals. After confirming a relationship between the estimated Vd values and serum inulin concentrations, we obtained an equation for calculating the estimated Vd values from serum inulin concentration 60 minutes postinjection. Finally, the GFR using a single-blood-sample method was obtained by substituting the dose of inulin injected, serum inulin concentration, sampling time, and estimated Vd value of each animal into Jacobsson's formula[7] again.


Six clinically healthy purpose-bred male cats and 3 CKD cats (2 spayed females and 1 castrated male) admitted to the Veterinary Teaching Hospital at Iwate University (IUVTH, Morioka, Japan) were used. The healthy cats were owned by IUVTH, and they were regarded as “healthy cats” from the results of clinical observations, hematology, serum chemistry, and urinalysis. CKD cats were used after obtaining the owners' informed consents for their participation in this investigation. The mean age of cats was 2.7 years (2–3 years) for healthy cats and 9.7 years for CKD cats. The mean body weight was 4.87 kg for healthy cats and 3.0 kg for CKD cats. The CKD cats were defined when diagnosed with over stage 2 (>1.60 mg/dL in serum creatinine concentration, 2 cats in the stage 2 and 1 cat in the stage 3) based on IRIS staging system by International Renal Interest Society[9] after 6-month to 1-year observation periods. All procedures were performed in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science[10] and approved by the Animal Experimental Ethics Committee of Iwate University (reference number A201026).

Inulin Administration and Blood Sampling

The number of cats used in the following studies is given in Supplemental Table 1.

To select the appropriate dose to measure GFR, inulin1 was administered in healthy cats according to a 3 × 3 Latin square design at doses of 30, 50, and 70 mg/kg, based on the results of a previous feline report.[11] Inulin (from chicory) was injected by an intravenous bolus into the cephalic vein through a 24-G-indwelling catheter.1 Blood (0.8 mL) was collected from the contralateral cephalic vein at predose and 30, 60, 90, and 120 minutes after each injection of inulin using a 25-G needle. The interval between each kinetics (30, 50, and 70 mg/kg) was at least 5 days.

To obtain a reference GFR by a 7-sample method, inulin was administered intravenously at 50 mg/kg to healthy cats (n = 4). Blood was collected at predose, and 5, 15, 30, 60, 90, 120, and 150 minutes later by the aforementioned method. The GFR values were calculated using a 1- or 2-compartment model.

To determine the proper blood-sample times, GFR was calculated by various combinations of blood sampling points on the basis of the data from the above reference GFR study. As serum inulin disappearance showed linearity 30–120 minutes after inulin injection, the combination of the sampling times for GFR estimations using the 1-compartment model was as follows: (a) 30, 60, 90, and 120 minutes later; (b) 30, 60, and 90 minutes later; (c) 30, 60, and 120 minutes later; (d) 30, 90, and 120 minutes later; and (e) 60, 90, and 120 minutes later. As the inulin concentration at 150 minutes was around the marginal level (20 μg/mL), its point was excluded from these analyses.


Serum inulin concentrations were colorimetrically determined by an autoanalyzer method using a commercially available kit.2 The limit of quantitation in serum inulin concentrations was 20 μg/mL, and the linearity was seen between 25 and 250 μg/mL under the present conditions. Validation studies revealed no significant difference between serum and plasma inulin concentrations, and the intra-assay coefficient of variability (CV) in serum inulin determination in cats was 5% (10 samples from pooled serum).

Blood urea nitrogen (BUN) and creatinine concentrations in sera were measured with the autoanalyzer3 on the same days that GFR was estimated.

Calculation of GFR

In the multisample (3-, 4-, or 7-sample) method using inulin, clearance calculations were based on the 1- or 2-compartment model. In brief, the area under the serum inulin concentration versus time curve (AUC) was calculated by the linear trapezoidal rule with extrapolation to infinity using the last 3 sample points in serum, and the clearance value (Cl) was calculated from the following formula.[7]

display math(1)

where Dose is the dose of inulin injected.

To determine serum inulin clearance because of the single-blood-sample method, the estimated volume of distribution (Vd1) of inulin in each animal was back-calculated by substituting Cl values and serum inulin concentrations (Ct) at 60, 90, or 120 minutes (t) obtained from the 3-sample method into the following Jacobsson's formula.[7]

display math(2)

The above formula can be transformed to the following equation by the classic Newton method,[12, 13] and the variable “b” value can also be solved by the same method.

display math(3)

The Vd1 value obtained was then reconfirmed by use of a commercially available spreadsheet software.4 To examine the Vd1 in each animal, an equation between the Vd1 values and C60 minutes, C90 minutes, or C120 minutes was determined by a scatter plot. Finally, the GFR value by the single-blood-sample method with inulin was measured by substituting the dose of inulin (Dose, 50 mg/kg), serum inulin concentration (C60 minutes) at 60 minutes (t), and estimated Vd (=Vd1) value calculated from each cat into the Jacobsson's formula (Equation (2)). The Cl term was regarded as the GFR for the present work.

The GFR is represented as mL/min/m2 based on the following body surface area (BSA)[13] partially with the indexation to bodyweight (mL/min/kg), because of a large variation between healthy and CKD cats in the body weight of cats used (Supplemental Table 1).

BSA = 0.10 × bodyweight (in kg)2/3

Statistical Analysis

Quantitative data are expressed as the mean ± standard deviation (SD) of each group. Comparison of GFR values between the 2 methods was performed according to standard recommendations for comparing analytical techniques based on Deming's regression[14] and Bland–Altman bias presentation[15, 16] using Prism 5.5


Appropriate Dose with Blood-Sample Times of Inulin to Estimate GFR

In clinically healthy cats given 30, 50, or 70 mg/kg inulin, mean inulin concentrations in serum disappeared with a semilogarithmic linearity from 30 to 120 minutes later at doses of 50 mg/kg or more and by 90 minutes later at 30 mg/kg (Fig 1A). Considering the detection sensitivity and minimum exposure of the whole body to inulin, a dose of 50 mg/kg was chosen (Fig 1B). At 50 mg/kg, a statistically significant difference (P < .01) was noted between GFR values estimated from the 1-compartment model using 4 blood-sample points (56.0 ± 5.4 mL/min/m2 converting to 3.30 ± 0.33 mL/min/kg) versus the 2-compartment model using 7 blood-sample points (47.3 ± 7.2 mL/min/m2 converting to 2.90 ± 0.44 mL/min/kg). No difference was seen between GFR values from 4 versus 3 blood-sample points (56.7 ± 4.9 mL/min/m2 converting to 3.40 ± 0.28 mL/min/kg) in the 1-compartment model (Fig 1C). In subsequent investigations, therefore, a combination of 50 mg/kg inulin with blood-sample times of 60, 90, and 120 minutes later was selected for a 3-sample method, and the 50 mg/kg dose was also chosen for a single-blood-sample method.

Figure 1.

Serum disappearance curve in a semilogarithmic plot and glomerular filtration rate (GFR) value determined by the combination of various blood sampling times in healthy cats. (A) Mean inulin disappearance curve in serum of cats intravenously given 30 (closed triangles), 50 (open circles), or 70 mg/kg inulin (closed diamonds), n = 3. (B) Representative inulin disappearance curve in serum of a cat intravenously given 50 mg/kg inulin. (C) GFR measured by combinations of (a) 30, 60, 90, and 120 minutes later, (b) 60, 90, and 120 minutes later (gray box), (c) 30, 60, and 90 minutes later, (d) 30, 90, and 120 minutes later, and (e) 30, 60, and 120 minutes later. Medians and quartiles are displayed in the box. Upper and lower bars represent maximum and minimum values, respectively, n = 4.

Estimation of the GFR by the Single-Blood-Sample Method Using Inulin

The equation for calculating the estimated Vd value was determined from a scatter diagram (Fig 2) as follows:

display math

where C is serum inulin concentration at 60 minutes, showing a close correlation (r = 0.93, P = .001, n = 9, sample number: 32, where the number elicits the sum of the GFR values collected from the same animals on different days). Serum inulin concentration 60 minutes later was chosen as it showed a somewhat high correlation between Vd1 values and serum inulin concentrations compared with that at 90 (r = 0.89, n = 9, P = .01, 30 samples) or 120 (r = 0.89, n = 9, P = .01, 34 samples) minutes.

Figure 2.

Correlation between the estimated Vd values and serum inulin concentrations 60 minutes after inulin injection using clinically healthy (closed circles) and CKD cats (open triangles). Vd = 485.7e−0.0148C, r = 0.93 (P < .001, n = 9, 32 samples).

A comparison of GFR obtained from the 3-sample method with that determined by the single-blood-sample method yielded a strong correlation (r = 0.99, P = .0001, n = 9, Fig 3A,B).

Figure 3.

Correlation between the glomerular filtration rate (GFR) values estimated by the 3-sample method (GFR inulinmulti) and single-blood-sample method (GFR inulinsingle 60 minutes) using inulin in healthy (closed circles) and CKD cats (open triangles), n = 9. (A) Scatter plot of GFR between the 2 methods. Deming's regression was y = 0.92x + 3.42, r = 0.99 (P < .0001). (B) Bland–Altman plot of the differences between the 2 methods. Mean bias (solid line): 0.54. The confidence limits (dotted line) of agreement plots are 95% (from −5.72 to 6.81).

Basal GFR Values Obtained by the Single-Blood-Sample Method Using Inulin

The basal GFR level in healthy cats was 52.8 ± 8.2 mL/min/m2 (converting to 2.95 ± 0.49 mL/min/kg, and, n = 6, 17 samples) ranging from 40.0 to 78.3 mL/min/m2 (converting to 2.23–4.37 mL/min/kg). Neither proteinuria nor abnormal specific gravity in urine was detected. During or after the administration of inulin, no adverse clinical signs were observed in any of the cats under the present protocols.


We assessed the validity of a single-blood-sample method using a bolus injection of inulin for estimating the feline GFR, in comparison with the conventional 3-sample method.

In healthy cats given 50 mg/kg inulin, a linear semilogarithmic plot of serum inulin concentrations versus time demonstrated the suitability of using a 1-compartment model for GFR calculation (3-sample method). Although the 1-compartment model was a simplification and only applied after an equilibration period (30–120 minutes after inulin injection), it was thought to underestimate AUC, compared with the 2-compartment model. Thus, the difference between GFR values estimated from the 1-compartment model using 4 blood-sample points and the 2-compartment model using 7 blood-sample points was considered to be caused by a difference in calculated AUC. Briefly, the AUC from the 1-compartment model was approximately 15% lower than that from the 2-compartment model, indicative of higher GFR in the former. However, no significant difference was detected between the GFR values estimated from 4 versus 3 blood sample collection times in the 1-compartment model. Based on these results, the approach involving 3 blood sample collection times (60, 90, and 120 minutes later) was chosen because of a minimum SD of difference in GFR values.

Under the conditions of this study, the Vd values ranged from 60 to 250 mL/kg and from 10 to 70 mL/kg in clinically healthy and CKD cats, respectively, although there was no report dealing with the Vd of inulin in the diseased states of cats so far. Moreover, the Jacobsson's formula for humans has not been validated for cats. Therefore, we focused on the formula described by Jacobsson,[7] including the inulin dose, Vd, and serum concentration of inulin concentration and sample collection time as variable factors.

Using the single-blood-sample method, the basal reference GFR values obtained in clinically healthy cats almost resembled the previously reported GFR data,[17, 18] although the experimental conditions and procedures were very different.

It has been reported that the formula derived for the GFR calculation with 1 blood sample requires that the Vd value be known, and its accuracy determines the accuracy in the method.[7] Likewise, when the Vd value of the tracer is known, the plasma disappearance curve can be closely approximated from a single, timed plasma measurement.[19] In our investigations, a relationship between the estimated Vd values and serum inulin concentrations was critical as a prerequisite for an equation to calculate the estimated Vd value. In the Deming's method, however, one of the assumptions was that both x and y (the result of the 2 methods) were subjected to random error, but in such a way that the ratio Var(x)/Var(y) was constant and not infinite or zero throughout the data range. When the variance of x was constant throughout the data range, the variance of y must be constant too.[20] Based on Bland and Altman bias presentation, all points obtained were within the agreement plots.

Jacobsson's formula can therefore be applied to cats, and the single-blood-sample method can be used for GFR estimation as an alternative to the multisample method. In contrast, because the GFR calculated from the estimated Vd value is based on many assumptions to predict the true GFR, and species differences have not been systematically evaluated in the formula from Jacobsson,[7] further studies are necessary to collect cumulative background data including various typed nephropathies.

In conclusion, the single-blood-sample method using inulin, instead of the 3-sample method, provides a practicable and ethical alternative for estimating glomerular filtration rate in cats.


We acknowledge Dr Yoji Furuhama and Mr Kanji Watanabe for providing information on the Newton method.

Conflict of Interest Declaration: Authors disclose no conflict of interest.


  1. 1

    Inulead, Fuji Yakuhin, Tokyo, Japan

  2. 2

    Dia-color-inulin, Toyobo, Osaka, Japan

  3. 3

    Toshiba Medical Systems, Tochigi, Japan

  4. 4

    Goal-Seek function of Microsoft Office Excel 2007, Microsoft, Tokyo, Japan

  5. 5

    GraphPad Software, San Diego, CA