Effects of broccoli extract on biodistribution and labeling blood components with 99 mTc-GH 1

PuRPOsE: People consume vegetables without the knowledge of the side effects of the biological and chemical contents and interactions between radiopharmaceuticals and herbal extract. To this end, current study is focused on the effects of broccoli extract on biodistribution of radiolabeled glucoheptonate (99mTc-GH) and radiolabeling of blood components. METHOds: GH was labeled with 99mTc. Quality control studies were done utilizing TLC method. Biodistribution studies were performed on male rats which were treated via gavage with either broccoli extract or SF as control group for 15 days. Blood samples were withdrawn from rats’ heart. Radiolabeling of blood constituents performed incubating with GH, SnCl2 and 99m Tc. REsuLTs: Radiochemical yield of 99mTc-GH is 98.46±1.48 % (n=8). Biodistribution studies have shown that according to the control, the treated group with broccoli has approximately 10 times less uptake in kidney. The percentage of the radioactivity ratios of the blood components is found to be same in both groups. CONCLusIONs: Although there is no considerable effect on the radiolabeling of blood components, there is an outstanding change on the biodistribution studies especially on kidneys. The knowledge of this change on kidney uptake may contribute to reduce the risk of misdiagnosis and/or repetition of the examinations in Nuclear Medicine.


Introduction
It's known that the consumption of natural foods which have high antioxidant capacity has protective effect on our health.
Depending on this knowledge nowadays there is a swift increase in consumption of these natural foods like vegetables and fruits.
In epidemiologic studies it's observed that there is an interaction between vegetable consumption and cancer prevention 1,2 .
Biological studies have shown that vegetables in Brassica family could reduce risks of the cancers, especially the gastrointestinal tract cancers 3 .Because of these and similar hypothesis, a great many of the vegetables in Brassica family have been subject of the chemical and biological studies.
Broccoli (Brassica oleracea Italica) from the Brassicaceae family is a nutrient source which contains many bioactive components including glucosinolates, flavonoids, minerals and antioxidants 4 .
In Nuclear Medicine, interactions between herbal extracts and radiopharmaceuticals are not completely understood.
Nevertheless patients consume these vegetables without the knowledge of the side effects of biological and/or chemical contents of these vegetables 5 .These contents may cause different variations on the behavior of radiopharmaceuticals.These variations may arise due to various factors including interactions and competition to binding site between herbal extract and radiotracers.It has been already reported that the biodistribution of radiopharmaceuticals used for imaging in Nuclear Medicine is also altered by herbal extracts 6,7 .In these studies, it has seen that some of the herbal extracts could alter uptake of the radiopharmaceuticals by organs, accordingly SPECT and PET scanning results could change.In some other studies it's concluded that drugs such as natural, herbal origin or synthetic drugs, as well as labeling conditions, could be effective on the labeling of blood constituents 8 .
Current study aims to evaluate in vitro and in vivo effects of broccoli extract on the biodistribution of technetium-99m labeled glucoheptonate ( 99m Tc-GH), which used for renal imaging and function testing; also known as 99m Tc glucoceptate.

Stability study of 99m Tc-GH
Stability of 99m Tc-GH was determined using TLRC method.99m Tc-GH was applied on ITLC-SG plates at 0 th , 1 st , 3 rd , 6 th , 7 th and 24 th hours.Then their radioactivities were counted.

Lipophilicity (LogP)
Tc-GH (0.100 mL) was added after 0.250 mL of n-octanol and 0.250 mL of phosphate buffer (pH 7) in a centrifuge tube.Then, this solution was vortexed for 1 min.and centrifuged for 30 min.at 2500 rpm.0.100 mL aliquots of each phase were taken and counted by Cd(Te) detector three times.LogP was calculated proportioning counts of phases.This experimental value of LogP was compared with theoretical LogP which was calculated ACD/ LogP software (Version 6.0 for Microsoft Windows).

Animals treatments
Male Wistar rats (n=16) were randomly divided into two groups as treated (n = 8) and control (n = 8) groups.In the treated group 330 mg/kg broccoli extract was administered to each rat by gavage during 15 days.Control group was treated with saline solution (0.9 % NaCl) by the same way and period.

Radiolabeling of blood components
Blood samples were withdrawn from rats' heart under anesthesia [xylazine and ketamine (75-100mg/kg+5-10g/kg)] and the rats were not sacrificed.0.500 mL of blood samples was mixed with 0.500 mL (1 mg/mL) GH and 0.010 mL (1 mg/mL) SnCl 2 and incubated 10 min.. Then 1 mCi (37 MBq) 99m Tc was added and incubated 10 min..These samples were centrifuged and the components [serum (S) -blood cells (BC)] separated.The samples was counted by Cd(Te) detector and the percentage of radioactivity was calculated.The data were expressed as mean ± standard deviation of the percentage of radioactivity (Table 3).

Biodistribution of 99m Tc-GH
At the end of 15 days, 99m Tc-GH (0.200 mL) was injected into the tail vein of the rats.The rats were euthanized by injection of sodium pentobarbital (200 mg/kg) by intraperitoneal via.After euthanasia, the organs were isolated and weighed (heart, lung, liver, kidney, small intestine, large intestine, stomach, spleen, pancreas, muscle, head, fat, thyroid, bladder, blood, testis, prostate and bone) and counted by Cd(Te) detector.The percentage of injected dose per gram of tissue weight (% ID/g) was determined.

Statistical analysis
Differences in the mean values of the measured activities were evaluated statistically by the SPSS 13 program (Univariate Variance Analyses and Pearson Correlation).Probability values <0.05 were considered as significant.Pearson correlation was carried out among different organs for 99m Tc-GH.

Results
Quality control studies of broccoli extract and GH were performed by HPLC and TLC.The relevant chromatograms were given in Figures 1 and 2.
According to HPLC chromatogram of broccoli extract, retention time (R t ) values of broccoli extract were 3.00, 11.50, 14.50 respectively.Lin et al. 3 studied on identification of phenolic components of collard greens, kale and Chinese broccoli, when we have compared with LC chromatograms of current study we have concluded that they were similar R t values according to HPLC chromatogram of broccoli extract (Figure 1).HPLC chromatogram of GH can be seen in Figure 2 and R t value of the GH was 2.60.Table 1 was shown R f values of broccoli extract and GH by TLC methods.The results of HPLC and TLC analysis were compatible with each other.

TABLE 1 -R f values of broccoli extract and GH by TLC methods.
High radiochemical yield [98.46 ±1.48 % (n=8)] was obtained by using TLRC method in SF solvent system.HPLRC and TLRC chromatograms were supported each other.As can be seen in Figure 4, stability studies showed that approximately 95% of 99m Tc-GH existed as an intact complex within 24 hours.Injected dose/g organ (%ID/g organ) of 99m Tc-GH for Wistar albino rats are given in Figure 5.The treatment with

discussion
The use of natural food has increased in all over the world.Natural foods are used for the human being however several biological effects and the consequences for the health have not been well established yet 9 .Contrary to the numerous theoretical and practical studies of the conventionally used pharmacologically active agents, data concerning interaction of diagnostic agents, including radiopharmaceuticals, with popular medicines are relatively scarce.There are lots of researches on herbal extract interaction with radiopharmaceuticals 10,11 .
One of these studies was done by using cauliflower which is in the same family with broccoli.In this study the rats were treated with cauliflower extract and SF, blood samples withdrawn from the treated group and control group were labeled with 99m Tc and there is no modification labeling red blood cells (RBC) with 99m Tc in the presence of the extract 12 .This result supports the results obtained in our experiment.Similarly, extracts of Pfaffia sp. 9 and Passiflora edulis f.Flavicarpa 13 has also not decreased the labeling of blood components with 99m Tc.However, it is reported that extracts of Fucus vesiculosus 10 and Cinnamomum zeylanicum 6 are decreased the labeling yield of blood components with 99m Tc.
In addition, the study which was done using extract of guarana and 99m Tc-DMSA, extract was reduced the labeling yield of RBC 11 .
All of these studies are shown that herbal extract can affect radiolabeling of blood cells and bioavailability of radiobiocomplexes.Labeling process of blood cells depends on the presence of a reducing agent and stannous chloride (SnCl 2 ) is utilized with this purpose 14 .In these processes labeling depends on interaction between 99m Tc and stannous chloride, this interaction could be influenced from the conditions of human plasma and its components 8 .
There are many studies about effects of herbal extracts on biodistribution of radiopharmaceuticals 7,13,15 .One of them is about the effect of the P. flavicarpa extract on the biodistribution of sodium 99m Tc (%ATI/g) in Wistar rats.In this study, it is found that P. flavicarpa treatment induced a significant (p<0.05)alteration on the %ATI/g of tissues from duodenum, spleen, pancreas, stomach as well as blood.No alteration on the %ATI/g of tissues from brain, liver, heart, kidney, lung, bone, muscle, thyroid and testis 13 .
In another study, it is shown that the mint (Mentha crispa) extract increased fixation of this radiobiocomplex in the pancreas, kidney, spleen, liver, and thyroid, there is no significant alteration in other organs.The reason of this alteration was tried to explain that as the radiobiocomplex studied is an ion, the effect of the components of extract would also act in the transport of the pertechnetate ion through the cellular membrane of determined organs 15 .Rocha and coworkers evaluated effects of sucralose sweetener sodium pertechnetate (Na 99m TcO 4 ) and diethylenetriamine pentaacetic acid labeled with 99m Tc ( 99m Tc-DTPA) biodistribution in rats.Sucralose sweetener was capable of altering %ATI/g of the radiopharmaceuticals in different organs 7 .
In literature, the changes at the biodistribution studies are explained by the presence of specific chemical compounds in the extract or by the generation of active metabolites capable to interfere with the biodistribution of the studied radiobiocomplex 15 .

Conclusions
Current study is an addition to other studies about herbal extract interaction with radiopharmaceuticals. Misdiagnosis and repetition of the examinations are due to these unknown interactions.
If herbal extract interaction with radiopharmaceuticals is known, imaging efficiency will be conclusively and the unnecessary radiation dose which is taken by patients with repeated imaging will reduce.Consequently we can put more reliable diagnosis.
broccoli extract significantly (p<0.05)modify the % ID/g of in particular kidney for 99m Tc-GH was decreased from 43.37±4.98 to 4.47±1.49.No significant alteration on the % ID/g of tissues from heart, lung, liver, small and large intestine, stomach, spleen, brain, thyroid, testis, prostate and bone.

TABLE 2 -
R f values of components by TLRC

TABLE 3 -
The percentage of radioactivity for P and RBC.