Establishment of Tissue Biodistribution and Blood Clearance Rates of Intravenously Administered Radioactive ⁵¹Cr³⁺ in New Zealand White Rabbits

Abstract

Radioactive trivalent chromium (⁵¹Cr³⁺) is a known radiopharmaceutical used to tag plasma proteins, platelets and also for estimation of blood volume. Nevertheless, there exist insufficient reports with limited sample sizes concerning its clearance from blood and its biodistribution after intravenous administration. This study focused to understand clearance rate of ⁵¹Cr³⁺ from blood and analyze its biodistribution. For biodistribution, six adult New Zealand white albino rabbits were injected with ⁵¹Cr³⁺ through their marginal vein. Percentage clearance of ⁵¹Cr³⁺ from blood was calculated by recording radioactive counts obtained at 1, 58, 61, 120, 180 and 240 minutes post-administration in thirty-three adult New Zealand white albino rabbits. For evaluating ⁵¹Cr³⁺ biodistribution, organs were surgically removed from the rabbits and weighed. Radioactivity of the organs and urine were counted in a nucleonix gamma-ray spectrometer with a NaI scintillation detector. Data were expressed as cps/g. Average clearance of ⁵¹Cr³⁺ was 34% from the first to the 58 minute. Subsequent measurements for hourly clearance at 120, 180 and 240 minutes showed percentage reduction of radioactivity of 33, 14 and 8, respectively. Minimal specific activities were found in the muscle and brain. Spleen, lungs, liver and kidneys exhibited moderate radioactivity. Urine tracer-concentrations were found to be ten times more than that of plasma. From this study, it has been observed that clearance of ⁵¹Cr³⁺ from blood was faster initially which slowed down progressively and there displayed moderate uptake of ⁵¹Cr³⁺ by certain organs. Understanding pharmacokinetics of ⁵¹Cr³⁺ is relevant for its potential use as a diagnostic tool.

Keywords:
Trivalent chromium; Biodistribution; blood volume; New Zealand white rabbits; blood clearance.

HIGHLIGHTS

• Tissue biodistribution of intravenously administered trivalent chromium.

• Blood clearance rates of intravenously administered trivalent chromium.

• Faster systemic blood clearance is through renal excretion.

• Understanding of the above parameters is relevant for its diagnostic applications.

HIGHLIGHTS

• Tissue biodistribution of intravenously administered trivalent chromium.

• Blood clearance rates of intravenously administered trivalent chromium.

• Faster systemic blood clearance is through renal excretion.

• Understanding of the above parameters is relevant for its diagnostic applications.

INTRODUCTION

The utility of radioactive chromium for plasma volume measurements was recognized as early as 1950 [¹]. Both, hexavalent and trivalent forms of chromium (⁵¹Cr³⁺) were employed as radiotracers for simultaneous clinical measurements of total red blood cell mass and plasma volume, in man [²]. It was observed that red cells did not imbibe ⁵¹Cr³⁺ following its intravenous (IV) injection; instead, ⁵¹Cr³⁺ had entirely become bound to the plasma proteins [¹, ²]. This property of ⁵¹Cr³⁺ permitted its utility for the measurement of plasma volume [³]. These observations eventually paved the way to determine blood volume.

There exist reports of ⁵¹Cr³⁺ being used for direct blood volume measurement, check the intactness of the blood-brain barrier, brain tumor localization and quantifying gastrointestinal protein loss [⁴-⁷]. However, a detailed evaluation of tissue biodistribution and tissue-toxicity-profiling of ⁵¹Cr³⁺ is obligatory before ascertaining its utility and safety in clinical practice. There are only a few articles that confer information for the same which are based on experiments in rats, dogs, sheep and rabbits without appropriate mentioning of the species of the animals. Another drawback observed was that all these studies were of smaller sample sizes that involved multiple subgroups, providing less reliability to the results [¹, ⁸-¹⁰].

Earlier, we had reported a novel method for the measurement of blood volume in rabbits which required the aid of a correction factor. To estimate the correction factor critical parameters like blood clearance, organ biodistribution and renal excretion rate were determined [⁴, ¹¹].

In this article, we report data on the tissue biodistribution and blood clearance rates of intravenously administered radioactive ⁵¹Cr³⁺ in New Zealand white rabbits which was previously unreported during the studies on blood volume determination using ⁵¹Cr³⁺.

MATERIAL AND METHODS

The institutional animal ethics committee approved the study. A total of thirty-three New Zealand white rabbits (Oryctolagus cuniculus) which include twenty-two females and eleven males weighing approximately 1.5 - 3.1 kg were used for the understanding of clearance of ⁵¹Cr³⁺ from the blood. Out of the thirty-three, six New Zealand white rabbits, three males and three females, were used for the biodistribution part of the study. All rabbits were fed with food and water ad libitum. During the experimental procedure, rabbits were kept on dietary restriction to prevent any fluctuations in blood volume. Rabbits were placed in a rabbit restrainer and the sites for injection and blood collection on both ears were prepared by shaving and wiping the spots with 70% alcohol. Ten minutes prior to the experiment, rabbits were anesthetized with ketamine hydrochloride injection (25 mg/kg body weight) through the marginal ear vein.

A 26G intravenous cannula (BD Neoflon^TM) was inserted into the marginal ear vein of the anesthetized rabbits and secured with adhesive plaster. Drugs and the radiopharmaceutical were injected through this route. Into the auricular artery of the other ear of the rabbits, a 24G intravenous cannula (BD Neoflon^TM) was inserted which was then secured with adhesive plaster. Blood was drawn out from this cannula [¹²].

Two mCi of ⁵¹Cr in aqueous solution was obtained from BRIT, DAE and GOI. Two syringes were loaded, both with one mL of solution that contained ⁵¹Cr and freshly prepared ascorbic acid (Sisco) (mass concentration of ascorbic acid = 2mg/mL). A two-hour holding time was given to ensure the complete reduction of chromium to ⁵¹Cr³⁺ [¹³]. Radioactivity in the loaded syringes was measured in a calibrated gamma-ray spectrometer with thallium-doped sodium-iodide (NaI (TI)) well-type scintillation detector coupled to a single-channel analyzer. All records of radioactivity were counted for thirty seconds and further converted to counts per second (cps) for calculations.

The clearance rate of ⁵¹Cr³⁺ from blood at the first minute to 58th minute following initial dose

Approximately 3000 cps of ⁵¹Cr³⁺ in one mL was injected through the IV cannula in the marginal vein which was followed by a 0.5 mL normal saline. Thereafter, residual activity was measured from this syringe. The injected ⁵¹Cr³⁺ was calculated after deducing the left-over activity in the loaded syringe. One minute subsequent to injection, one mL blood sample was collected from the IV cannula in the auricular artery. This sample was obtained for blood volume calculation. At the 58^th minute following ⁵¹Cr³⁺ injection, another 1 mL blood sample was drawn and its radioactivity measured. The percentage clearance of ⁵¹Cr³⁺ was calculated using the following formula:

$$\frac{cps1-cps58}{cps1}x100$$

cps1 = counts obtained from one mL blood sample drawn at one minute post ⁵¹Cr³⁺ injection.

cps58 = counts obtained from one mL blood sample drawn at 58th minute post ⁵¹Cr³⁺ injection.

The clearance rate of ⁵¹Cr³⁺ from blood following a spike dose

Ensuing the above procedure, a repeat/second dose of approximately 3150 cps of ⁵¹Cr³⁺ was injected through the marginal vein followed by a 0.5 mL saline wash at the 60th minute. After this spiking dose, one mL blood sample was obtained at the 61st minute to measure the radioactivity. This was followed by collection of one mL blood sample each at 120, 180 and 240 minutes post first dose of ⁵¹Cr³⁺ injection for radioactivity measurement, in cps/mL, for calculation of clearance kinetics. Percentage clearance was obtained using the same formula mentioned above.

Biodistribution study of ⁵¹Cr³⁺ in organs of sacrificed rabbits

At the end of four hours post ⁵¹Cr³⁺ injection, six rabbits (three males and three females) were euthanized by an overdose administration of ketamine hydrochloride intravenously. The organs were removed surgically and weighed in a pre-weighed container. A small section was cut from each of the organs and was placed in a pre-weighed 1.5 mL microcentrifuge tube. The organ-section was then weighed and its radioactivity measured.

The bladder was completely drained and the urine weighed. One mL urine sample, measured by weight, was obtained from the total volume of urine.

Radioactivity of different organs as well as urine was counted and the data was expressed as cps/g of tissue using the following formula:

$$cpspergoftissue=\frac{countsperg}{360}$$

RESULTS

The amount of ⁵¹Cr³⁺ in blood decreased over time post its intravenous administration. The average clearance of ⁵¹Cr³⁺ was 34% from the first to the 58th minute. Clearance of ⁵¹Cr³⁺ from blood after the first dose is displayed in Figure 1.

Figure 1
Clearance rate of ⁵¹Cr³⁺ from blood after intravenous administration of initial dose.

Figure 2 exhibits increase in radioactivity-counts by 1.72 times in blood owing to the spike dose of ⁵¹Cr³⁺ at the 60^th minute post first dose. Subsequent measurements of counts for hourly clearance from blood at 120, 180 and 240 minutes indicated a percentage reduction of radioactive-counts of 33, 14 and 8, respectively. Counts are expressed in cps/mL. With time there appeared a reduction in rate of clearance of ⁵¹Cr³⁺.

Figure 2
Clearance rate of ⁵¹Cr³⁺ from blood following intravenous administration of spike dose.

Radioactivity in the different organs was measured at the 240^th minute (four hours) following the first dose of ⁵¹Cr³⁺injection. This measurement reflects the cumulative effects of both the first as well as the spike doses, amounting to approximately 6150 cps. The data belonging to the parametric type was expressed in mean ± SD and in median and inter quartile range (IQR) for those measurements that obeyed the non-parametric pattern. The radioactivity counts of all organs were observed to be lesser than that of blood. Minimal specific activities were exhibited by the muscle and brain tissues. Tissues of the heart, bone, lymph node, diaphragm, stomach, ovaries, pancreas, epididymis, submandibular gland and testis retained diminutive amounts whereas the spleen, lungs, liver and kidneys presented moderate radioactivity. Maximum radioactivity was obtained in the urine samples. Urinary tracer concentrations were found to be exceeding ten times that seen in plasma. Table 1 shows the estimated counts in the various tissues.

Table 1
Distribution of ⁵¹Cr (III) after intravenous injection.

Organs	No. of rabbits	cps/g of tissue (Mean ± SD)
Blood	5	11.84±3.69
Heart #	6	3.07(2.40)
Spleen #	6	5.94(14.49)
Lymph node #	5	3.72(1.99)
Lungs #	6	7.84(7.76)
Diaphragm #	5	1.11(1.52)
Muscle	5	0.92±0.34
Bones	6	3.23±1.13
Brain #	5	0.27(0.17)
Liver #	6	6.15(1.61)
Pancreas #	3	1.95(1.26)
Stomach #	5	0.75(4.72)
Submandibular gland	3	1.69±0.82
Kidneys	6	8.71±4.17
Urine	5	139.30±23.40
Testis	2	1.8(0.87)
Epididymis	3	2.79±0.95
Ovaries #	3	0.47(4.50)

# indicates Median & IQR

DISCUSSION

Clearance of ⁵¹Cr³⁺ from circulation was observed at multiple time-points following the administration of two doses of ⁵¹Cr³⁺. The percentage clearance of ⁵¹Cr³⁺ that was observed at the 58th minute, subsequent to the first dose of approximately 3000cps of ⁵¹Cr³⁺, was 34%. Owing to its high affinity to form colloid complexes in plasma, levels of ⁵¹Cr³⁺ were found to be high in blood [³]. Following the spike dose administration of ⁵¹Cr³⁺ at the 60th minute following the first dose, percentage clearance of ⁵¹Cr³⁺ observed at 61, 120, 180 and 240 minute appeared to be diminishing with values 33,14 and 8, respectively. This tapering clearance of ⁵¹Cr³⁺ attributes to the flux of the tracer across capillaries, its loss through urine, its excretion through the gut and also its flux out of the tissues. Kraintz and Talmage described in their study the determination of radioactivity distribution in rabbits following intravenous administration, wherein it was suggested that the gradual and decreased clearance of ⁵¹Cr³⁺ occurred due to its deposition in the reticuloendothelial system [⁹]. In our study, it is clear that the clearance of dual-dosed ⁵¹Cr³⁺ from blood is independent of the number of dose administered. Similar studies on rats showed that the faster initial clearance is attributed to the loss of tracer through urine whereas the slow clearance observed in the later hours is due to the reduced release of chromium from the reticuloendothelial system [⁸]. This study correspondingly reports a rapid clearance rate of chromium in the initial hours that diminishes in the later hours.

In addition to determining the circulatory ⁵¹Cr³⁺ clearance rate, we also attempted to investigate the biodistribution of ⁵¹Cr³⁺ in various organs, four hours post ⁵¹Cr³⁺ intravenous administration of two doses, which totally amounted to about 6150 cps. It appeared that different organs exhibited varying amounts of ⁵¹Cr³⁺ retention. However, radioactivity measured in each tissue was lesser when compared to that obtained from blood [⁵]. Moderate ⁵¹Cr³⁺ uptake was exhibited by the spleen, lungs, liver and kidneys. Minimal activity was presented by the muscle and brain attributable to the inability of ⁵¹Cr³⁺ to penetrate the muscle membrane and the blood-brain barrier respectively [⁵]. Tissues of the heart, bone, lymph nodes, diaphragm, stomach, ovaries, pancreas, epididymis, submandibular gland and testis retained a minute amount of radioactivity. The study conducted by Hopkins indicated a higher ⁵¹Cr³⁺ uptake by mature testis of rats. On the contrary, our results in rabbits did not show significant ⁵¹Cr³⁺ uptake [⁸]. Urinary tracer concentrations were found to be ten times more than that recorded from the plasma. In a similar study, Edstrom observed that one-third of the dose given intravenously to rabbits was excreted in three days [⁵]. There are limited studies that describe biodistribution using ⁵¹Cr³⁺ in rabbits [⁹,¹⁰]. Also, existing studies fail to mention the type of rabbit species involved in the experiments. This makes it challenging to rely on available data for initiating newer studies. Moreover, a proper comparison of data is difficult to achieve.

An unexpected downfall we encountered during the procedure was that there occurred radioactive contamination among few organs during the dissection process. Such organs were discarded and not included in the study and hence a disparity in the number of organs harvested. Repeat experimentation on fresh rabbits to equalize the sample size was not possible due to ethical constraints. Therefore, in future studies, we advise that such issues like contamination of organs during dissection along with animal attrition have to be looked into while planning experiments on biodistribution priorly.

CONCLUSION

Understanding the pharmacokinetics of ⁵¹Cr³⁺ is relevant for its potential use as a diagnostic tool. ⁵¹Cr³⁺ has gained popularity in experimental research in fields of blood volume measurements and in experiments that required to verify the intactness of membrane barriers. However, a proper comprehension of its biodistribution is necessary for its utility in the medical domain. This study establishes specific details regarding it’s clearance that was deciphered in real-time and also displays its biodistribution.

⁵¹Cr³⁺ clearance from blood occurred at a biphasic manner, exhibiting rapid clearance initially and that slowed down progressively. Moderate uptake of ⁵¹Cr³⁺ was presented by certain organs, especially by those that housed the reticuloendothelial system. Radioisotopes hold great promise in therapeutics and diagnostics provided its clearance and biodistribution are understood.

Acknowledgments:

We acknowledge the support extended by the department of nuclear medicine, Kasturba Medical College - Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India and Central Animal Research Facility, Manipal Academy of Higher Education, Manipal, Karnataka, India. We also thank Dr. Prakash P Y., Associate Professor, Department of Microbiology, Kasturba Medical College - Manipal, Manipal Academy of Higher Education for the guidance and support in drafting this manuscript.

• Funding: “This research received no external funding”

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Edited by

Editor-in-Chief: Paulo Vitor Farago

Associate Editor: Renata Marino Romano

Publication Dates

• Publication in this collection
  25 July 2022
• Date of issue
  2022

History

• Received
  27 Sept 2021
• Accepted
  22 Mar 2022