NL2032749B1 - Method for determining absolute oral bioavailability of mercury in mercury sulfide or mercury sulfide-containing substances by mercury radioisotope - Google Patents
Method for determining absolute oral bioavailability of mercury in mercury sulfide or mercury sulfide-containing substances by mercury radioisotope Download PDFInfo
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- 229910052753 mercury Inorganic materials 0.000 title claims abstract description 144
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 title claims abstract description 103
- QXKXDIKCIPXUPL-UHFFFAOYSA-N sulfanylidenemercury Chemical compound [Hg]=S QXKXDIKCIPXUPL-UHFFFAOYSA-N 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000000126 substance Substances 0.000 title claims abstract description 24
- 210000002700 urine Anatomy 0.000 claims abstract description 107
- 230000005865 ionizing radiation Effects 0.000 claims description 159
- 229920002472 Starch Polymers 0.000 claims description 146
- 239000008107 starch Substances 0.000 claims description 146
- 235000019698 starch Nutrition 0.000 claims description 129
- 241001465754 Metazoa Species 0.000 claims description 126
- 239000000243 solution Substances 0.000 claims description 104
- 239000000725 suspension Substances 0.000 claims description 77
- 238000010171 animal model Methods 0.000 claims description 65
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 50
- 239000002253 acid Substances 0.000 claims description 34
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- 238000005406 washing Methods 0.000 claims description 29
- 230000000717 retained effect Effects 0.000 claims description 25
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- 238000012360 testing method Methods 0.000 claims description 16
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- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 claims description 5
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- 229960000443 hydrochloric acid Drugs 0.000 description 22
- 235000011167 hydrochloric acid Nutrition 0.000 description 22
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(II) oxide Inorganic materials [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 20
- 239000003814 drug Substances 0.000 description 19
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- 239000000843 powder Substances 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 12
- JJWSNOOGIUMOEE-UHFFFAOYSA-N Monomethylmercury Chemical compound [Hg]C JJWSNOOGIUMOEE-UHFFFAOYSA-N 0.000 description 11
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 10
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- 230000027939 micturition Effects 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 229910052979 sodium sulfide Inorganic materials 0.000 description 4
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 4
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 4
- 229940001474 sodium thiosulfate Drugs 0.000 description 4
- 235000019345 sodium thiosulphate Nutrition 0.000 description 4
- 229940048910 thiosulfate Drugs 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
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- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
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- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 3
- -1 methyl mercury) Chemical compound 0.000 description 3
- 229920001592 potato starch Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000478345 Afer Species 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
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- 229960002523 mercuric chloride Drugs 0.000 description 2
- 229940100892 mercury compound Drugs 0.000 description 2
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- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
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- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
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- HDWIHXWEUNVBIY-UHFFFAOYSA-N bendroflumethiazidum Chemical compound C1=C(C(F)(F)F)C(S(=O)(=O)N)=CC(S(N2)(=O)=O)=C1NC2CC1=CC=CC=C1 HDWIHXWEUNVBIY-UHFFFAOYSA-N 0.000 description 1
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- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
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- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
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- 150000002731 mercury compounds Chemical class 0.000 description 1
- 230000004148 metal metabolism Effects 0.000 description 1
- BABMCXWQNSQAOC-UHFFFAOYSA-M methylmercury chloride Chemical compound C[Hg]Cl BABMCXWQNSQAOC-UHFFFAOYSA-M 0.000 description 1
- ZVTVGJJRBKPPEW-UHFFFAOYSA-N methylmercury(1+);nitrate Chemical compound C[Hg]O[N+]([O-])=O ZVTVGJJRBKPPEW-UHFFFAOYSA-N 0.000 description 1
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- XLNZEKHULJKQBA-UHFFFAOYSA-N terbufos Chemical compound CCOP(=S)(OCC)SCSC(C)(C)C XLNZEKHULJKQBA-UHFFFAOYSA-N 0.000 description 1
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- 229960005088 urethane Drugs 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/16—Measuring radiation intensity
- G01T1/161—Applications in the field of nuclear medicine, e.g. in vivo counting
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/16—Measuring radiation intensity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/16—Measuring radiation intensity
- G01T1/20—Measuring radiation intensity with scintillation detectors
- G01T1/202—Measuring radiation intensity with scintillation detectors the detector being a crystal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/16—Measuring radiation intensity
- G01T1/28—Measuring radiation intensity with secondary-emission detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/01—Investigating materials by wave or particle radiation by radioactivity, nuclear decay
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Abstract
A method for determining an absolute oral bioavailability of mercury in mercury sulfide or a mercury sulfide—containing substance by a mercury radioisotope is provided. The present invention overcomes the interference technical barrier of mercury 5 contamination generated during the process of sample collection, pre—treatment and determination. Meanwhile, the present invention establishes a method for determining an absolute oral bioavailability (absorption rate) of mercury in mercury sulfide based on the amount of mercury retention in vivo and the amount of 10 mercury excreted via urine by labeling a sample to be tested with 203Hg (half—life is 46.612 days) via a highly sensitive v—ray counting method. The method will lay the foundation for the exposure risk assessment and toxicological and pharmacological studies of mercury in mercury sulfide and mercury sulfide 15 substances.
Description
METHOD FOR DETERMINING ABSOLUTE ORAL BIOAVAILABILITY OF MERCURY IN
MERCURY SULFIDE OR MERCURY SULFIDE-CONTAINING SUBSTANCES BY
MERCURY RADIOISOTOPE
The present invention relates to the technical field of bioa- vailability determination, in particular to a method for determin- ing an absolute oral bicavailability of mercury in mercury sulfide or a mercury sulfide-containing substance by a mercury radioiso- tope.
Mercury is a ubiquitous heavy metal element, and its chemical forms include elemental mercury, organic mercury (e.g., methyl mercury), soluble inorganic mercury {e.g., mercuric chloride) and insoluble inorganic mercury (e.g., mercuric sulfide). The insolu- ble mercuric sulfide has been used for the treatment of diseases in a variety of traditional medicines and has had a history of ap- plication for thousands of years, for example, in the application of cinnabar and numerous cinnabar formulations in traditional Chi- nese medicine, such as Angong Niuhuangwan pill, Xiaoer Jingfeng powder, compound aloe capsule, cinnabar sedative pill, semen bio- tae pulse-invigorating and heart-nourishing pill, and children's indigestion powder; Tsothay (Zuotai) and numerous Tsothay formula- tions in Tibetan medicine, such as Qishiweizhenzhu pill, Ren- gingchangjue, Rengingmangjue, Zuozhudaxi, Dangzuo powder and Er- shiwei Shanhu pill; Peltophorum tonkinense in Mongolian medicine and its compound Xiongyinsan powder, Xakuzhuer, 18-Ingredient
Menggenwusu pill, and the like; Kajjali, Ras-Sindoor and compound formulations thereof in Indian medicine. These mercury sulfide- containing medicaments are still clinically used in traditional
Chinese medicine frequently. However, it is believed in modern science that mercury is a toxic element and may cause a variety of damages to the body. It remains unclear whether and to what extent these mercury sulfide-containing medicaments pose an effective mercury exposure risk to the body.
The oral bicavailability (absorption rates) of elemental mer- cury, soluble inorganic mercury and organic mercury have been re- ported by the US Environmental Protection Agency (EPA), the World
Health Organization (WHO) and other organizations through studies.
The specific research status is as follows: (1) Oral bioavailability of metallic elemental mercury: EPA studies have reported that the amount of liquid metallic mercury element absorbed by the digestive tract is very limited; it has been found by Bornmann, et al., that the oral bicavailability of elemental mercury in rats is less than 0.01% (Bornmann, G., G.
Henke, H. Alfes, et al. 1970. Concerning the enteral absorption of metallic mercury. Arch. Toxicol. 26:203-209). (2) Oral bioavailability of soluble inorganic mercury: it has been reported by the studies of EPA and WHO that soluble inorganic mercury has a high bioavailability in the gastrointestinal tract.
It has been found by Rohla, et al. by radioactive **’Hg-labeled soluble inorganic mercury that the bioavailability of mercury in the human gastrointestinal tract is 7-15% (Miettinen, J.K. 1973.
Absorption and elimination of dietary (Hg'') and methyl mercury in man. In: Mercury, Mercurial, and Mercaptans, M.W. Miller and T.W.
Clarkson, Ed. Springfield, IL. p. 233-243; Rahola, T., T. Hattula,
A. Korolainen and J.K. Miettinen. 1973. Elimination of free and protein-bound ionic mercury in man. Ann. Clin. Res. 5:214-219). By the radioactive °%Hg-labeled method, it has been found in rats and mice by Kostial et al. and Nielsen et al. that the true oral bioa- vailability of soluble inorganic mercury from the gastrointestinal tract is close to 20%, but the result measured actually is slight- ly lower (Kostial, K., D. Kelle, S. Jugo, et al. 1978. Influence of age on metal metabolism and toxicity. Environ. Health Perspect. 25:81-86; Nielsen, J.B. 1992. Toxicokinetics of mercuric-chloride and methylmercuric chloride in mice. J. Toxicol. Environ. Health. 37(1):85-122). Piotroski, et al. have used ‘%Hg-labeled mercury chloride to study the absorption of Hg** in intestinal tract and to estimate that about 3-4% of mercury chloride is absorbed at a body weight of 0.2-12.5 mg/kg; about 8.5% of mercury chloride is ab- sorbed at a body weight of 17.5 mg/kg, and about 6.5% of mercury chloride is absorbed at a body weight of 20 mg/kg (Piotrowski JK,
Szymanska JA, Skrzypiúska-Gawrysiak M, Kotelo J, Sporny S. Intes- tinal absorption of inorganic mercury in rat. Pharmacol Toxicol. 1992 Jan; 70(1): 53-5. doi: 10.1111/j.1600-0773.1992.tb00426.x.
PMID: 1594537). (3) Oral bioavailability of methyl mercury: it has been re- ported by EPA and WHO studies that methyl mercury can be absorbed efficiently in intestinal tract. It has been found by Ablerg, et al. and Miettinen, et al. via radioactive *““’Hg-labeled methyl mer- cury that about 95% of the methyl mercury in the fish body can be absorbed by the gastrointestinal tract of the test subject (Aberg,
B., L. Ekman, R. Falk, U. Greitz, G. Persson and J. Snihs. 1969.
Metabolism of methyl mercury “Hg compounds in man: Excretion and distribution. Arch. Environ. Health 19:478-484; Miettinen, J.K. 1973. Absorption and elimination of dietary (Hg++) and methyl mer- cury in man. In: Mercury, Mercurial, and Mercaptans, M.W. Miller and T.W. Clarkson, Ed. Springfield, IL. p. 233-243). Moreover,
Aberg, et al. have used radioactive “’’Hg-labeled methylmercury ni- trate to study its absorption by the body. The result shows that more than 95% of methylmercury nitrate is absorbed by the body in aqueous solution (B., L. Ekman, R. Falk, U. Greitz, G. Persson and
J. Snihs. 1969. Metabolism of methyl mercury (203Hg) compounds in man: Excretion and distribution. Arch. Environ. Health 19:478- 484).
The research reports and literatures from EPA and WHO have indicated the oral bioavailability of elemental mercury, soluble inorganic mercury and organic mercury, but no bioavailability of mercury sulfide. Mercury sulfide is a typical extremely insoluble covalent sulfide; black B-HgS has a HgS solubility product (Ksp) of 1.6%107%%, and red «-HgS has a B-HgS Ksp of 4x10 (Lange's
Handbook of Chemistry (15th ed)). It has been indicated in the EPA research report (Hassett-Sipple, B, Swartout, J, and Schoeny, R.
Mercury study report to Congress. Volume 5. Health effects of mer- cury and mercury compounds. United States: N. p., 1997. Web.
Doi:10.2172/575119.; P U.S. EPA. Provisional Peer-Reviewed Toxzici- ty Values for Mercuric Sulfide. U.S. Environmental Protection
Agency, Washington, DC, EPA/690/R-02/011F, 2002.) and the WHO guideline (Guidance for identifying populations at risk from mer- cury exposure, WHO, 2008) that mercury sulfide is an insoluble compound, and mercury therein is poorly absorbed by the gastroin- testinal tract. Revis, et al., have administered 5 male mice in- tragastrically a single dose of radioactive *“*Hg-labeled mercury sulfide, and collected feces for 10 consecutive days, and then sacrificed the mice to take out the intestinal tract. Afterwards, the absorption rate of mercury sulfide was calculated by a reverse calculation method, specifically as follows: firstly, a difference between the total amount of mercury in mercury sulfide ingested via intragastric administration and the total amount of mercury in feces and intestinal tract was calculated, and then the total amount of mercury in mercury sulfide ingested via intragastric ad- ministration was divided by the difference; and the obtained value served as the oral absorption rate of mercury sulfide (oral ab- sorption rate of mercury sulfide = (total amount of mercury in mercury sulfide ingested via intragastric administration - total amount of mercury in feces and intestinal tract)/total amount of mercury in mercury sulfide ingested via intragastric administra- tion), specifically calculated as 0.4% (Revis, N.W., Osborne,
T.R., Holdsworth, G. et al. Mercury in soil: A method for as- sessing acceptable limits. Arch. Environ. Contam. Toxicol. 19, 221-226 (1990)). However, it has been indicated by the EPA's re- port (EPA/690/R-02/011F) that the oral absorption rate of mercury sulfide obtained using the reverse calculation method is not reli- able due to a very big error and thus, is not statistically sig- nificant. Specifically, the total radicactive intensity of mercury in mercury sulfide ingested via intragastric administration is 336,580 + 39,304 dpm (mean + SD); the total radioactive intensity of mercury in feces and intestinal tract is 335,276 + 46,498 dpm (mean + SD), and the oral absorption rate of mercury sulfide ob- tained by the difference between the two groups of data is 0.4%.
However, the standard deviation (SD) of these two groups of data is greater than 10% of the mean. Therefore, there is only 0.4% difference between these two groups of data, which is not statis- tically significant. Moreover, Jie Liu, et al., and the like have speculated that the oral bicavailability (absorption rate) of cin-
nabar (the major component is o-HgS) is less than 0.2% based on the literature reports in the review part (Liu J, Shi JZ, Yu IM,
Goyer RA, Waalkes MP. Mercury in traditional medicines: is cinna- bar toxicologically similar to common mercurials? Exp Biol Med 5 (Maywood). 2008 Jul;233(7):810-7. doi: 10.3181/0712-MR-336. Epub 2008 Apr 29. PMID: 18445765; PMCID: PMC2755212); and the value is indirectly calculated according to the fold difference of mercury concentration in mice organs between the mercuric chloride group and the cinnabar group. This bioavailability (absorption rate) value may be seriously overestimated based on the Ksp of red mer- cury sulfide (4x107%%)
To sum up, the bicavailability of mercury in orally exposed mercury sulfide and mercury sulfide-containing medicaments has been not clear at present. Moreover, there is lack of a reliable method for determining the gastrointestinal bicavailability of mercury suitable for mercury sulfide. Bicavailability is to re- flect a proportion of a given drug that enters the human body for circulation. Bioavailability describes the percentage of the amount of the drug which is absorbed by the gastrointestinal tract and reaches the systemic circulation blood after passing through the liver accounting for the total amount of drug taken orally.
Bicavailability is divided into an absolute bicavailability and a relative bioavailability. The absolute bioavailability is a ratio of the amount of drug absorbed into systemic circulation to the dosage of administration. In practical work, the absolute bioa- vailability is taken as the ratio of the blood concentration-time area under the curve (AUC) of extravascular administration to the blood concentration-time AUC of intravenous injection. Generally, the absolute bioavailability of intravenous formulations is con- sidered to be 100%. The relative bioavailability refers to the bi- oavailability obtained by comparing the degree and rate of absorp- tion between different formulations of the same drug. The absolute bioavailability is an irreplaceable important indicator to evalu- ate an exposure risk of mercury sulfide-bearing drugs and mercury sulfide in the environment. However, since mercury sulfide is a typical extremely insoluble compound, the method of the ratio of oral administration AUC to intravenous injection AUC is not suita-
ble for determining the absolute oral bicavailability. Therefore, how to accurately determine the absolute oral bicavailability of mercury in mercury sulfide and a mercury sulfide-containing sub- stance is a technology to be solved urgently at present.
The objective of the present invention is to provide a method for determining an absolute oral bicavailability of mercury in mercury sulfide or a mercury sulfide-containing substance by a mercury radioisotope. The method provided by the present invention can achieve the accurate determination of the absolute oral bioa- vailability of mercury in mercury sulfide or a mercury sulfide- containing substance, which lays the foundation for the exposure risk assessment and toxicological/pharmacological studies of mer- cury in mercury sulfide and traditional drugs containing mercury sulfide.
To achieve the above objective, the present invention pro- vides the following technical solutions:
The present invention provides a method for determining an absolute oral bioavailability of mercury in mercury sulfide or a mercury sulfide-containing material by a radioisotope *°Hg, in- cluding following steps of: mixing a gelatinized starch solution with a sample to be tested to obtain a pharmaceutical starch suspension; the sample to be tested is mercury sulfide or a mercury sulfide-containing sub- stance which is labeled by *%Hg; intragastrically administering the pharmaceutical starch sus- pension to experimental animals via a syringe as experimental group animals; intragastrically administering the gelatinized starch solution to experimental animals as blank control group an- imals; where before the intragastric administration, ionizing ra- diation counting of the total *%Hg in the pharmaceutical starch suspension or gelatinized starch solution of the syringe used for intragastric administration is determined; after the intragastric administration, ionizing radiation counting of the *“*Hg in the re- sidual pharmaceutical starch suspension or gelatinized starch so- lution in the syringe was determined;
collecting urine of the experimental group animals and blank control group animals after the intragastric administration re- spectively, then removing digestive tracts of the obtained experi- mental animals, and successively performing primary washing, im- mersion treatment with a mixed acid solution and secondary wash- ing, then collecting the remaining bodies of the obtained experi- mental animals; wherein the experimental animals were stimulated to urinate before intragastric administration and the obtained urine is discarded, and urine is collected continuously at a pre- set time interval after intragastric administration and before re- moving the digestive tracts; the mixed acid solution is a mixed solution of nitric acid and hydrochloric acid; respectively determining the collected urine and the remain- ing bodies of the experimental animals to obtain the ionizing ra- diation counting of *%Hg in the urine and the ionizing radiation counting of **%*Hg retained in the remaining bodies of the experi- mental animals, respectively; calculating the absolute oral bioavailability of mercury in the sample to be tested according to the formula shown in Formula
I: absolute oral bicavailability of mercury (3) = (Mohs) / (Na
Nss) x 100% Formula I; where, N,,-corrected zero-time ionizing radiation counting of the total “Hg retained in urine and remaining bodies of the ex- perimental group animals;
Mo-corrected zero-time ionizing radiation counting of the to- tal ‘Hg retained in urine and remaining bodies of the blank con- trol group animals;
No-corrected zero-time ionizing radiation counting of *“’Hg digested in the experimental group animals via intragastric admin- istration;
Nso-corrected zero-time ionizing radiation counting of **%Hg digested in the blank control group animals via intragastric ad- ministration; the Nao, Mo; Ns and Ny; are calculated according to the formu- las shown in Formula II, Formula III, Formula IV and Formula V, respectively:
Nao=Nao poay + Nao urine Formula IT;
Noo=Noo poay + Moo urine Formula III;
Neo=Neo pefore — Nog after Formula IV;
Nao=Nao before — Nao after Formula V; where, Nas pogy corrected zero-time ionizing radiation counting of the “Hg retained in the remaining bodies of the experimental group animals;
Nao wine corrected zero-time ionizing radiation counting of the 2%Hg in urine of the experimental group animals;
No vosy corrected zero-time ionizing radiation counting of the “Hg retained in the remaining bodies of the blank control group animals;
No wins corrected zero-time ionizing radiation counting of the “Hg in urine of the blank control group animals;
Ng petore Corrected zero-time ionizing radiation counting of the total *%Hg in the pharmaceutical starch suspension in the sy- ringe used before intragastric administration to the experimental group animals;
Nog after Corrected zero-time ionizing radiation counting of the “®"Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals;
Nao pefore—COrrected zero-time ionizing radiation counting of the total “Hg in the gelatinized starch solution in the syringe used before intragastric administration to the blank control group animals;
Nas after Corrected zero-time ionizing radiation counting of the 2%Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals; the Nas bodyr Nao uriner Ng body? Noa urine’ Neg beforer Nog afters Nao before and Ny after are zero-time ionizing radiation counting obtained by respectively correcting “Hg ionizing radiation counting Nat poays Nat urines Dot podyr Mot uriner Net petorer Net atverr Nst before and Na ater determined at t according to a “Hg decay correction formula; the Na voay is ionizing radiation counting of the Hg re- tained in the remaining bodies of the experimental group animals determined at t; and Nu urine is ionizing radiation counting of the ““*Hg retained in urine of the experimental group animals deter- mined at t; the Me nosy is ionizing radiation counting of the Hg re- tained in the remaining bodies of the blank control group animals determined at t; and Nx wine is ionizing radiation counting of the “Hg in urine of the blank control group animals determined at t; the Nu perore is ionizing radiation counting of the total *" Hg in the pharmaceutical starch suspension in the syringe used before intragastric administration to the experimental group animals de- termined at t, and Nu after is ionizing radiation counting of the “Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals determined at t; the Nu pareve is ionizing radiation counting of the total ‘Hg in the gelatinized starch solution in the syringe used before in- tragastric administration to the blank control group animals de- termined at t, and Nu after is ionizing radiation counting of the “Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals determined at t; the “Hg decay correction formula is shown in Formula VI:
N, = Nye™** Formula VI; where N.-ionizing radiation counting of the “Hg determined at t;
Ns-corrected zero-time ionizing radiation counting of the ‘VHg;
A-decay constant, and the A=In{(2})/t1:, ti: is a half-life of the “Hg; t-zero time refers to certain time point prior to the intra- gastric administration of the first experimental animal or the time point when the first experimental animal is administered in- tragastrically; and the t20 s.
Preferably, the method for preparing the ?%*Hg-labeled mercury sulfide includes following steps of: irradiating HgO with a neutron ray, and placing the irradiat- ed HgO such that the obtained ‘HgO decay weakens, thus obtaining dissolving the *°°Hg0O into an inorganic acid, and mixing the obtained “Hg*' solution with a precipitant solution for reaction, then successively washing and drying the obtained precipitate to obtain the °%Hg-labeled mercury sulfide; wherein the precipitant in the precipitant solution is a soluble sulfide or thiosulfate.
Preferably, the irradiation time is 2 one week; the placing time is Z 10 days.
Preferably, a molar ratio of the precipitant to ““Hgo is (1- 2:1.
Preferably, the *“’Hg-labeled mercury sulfide has a specific radioactivity of 1-20 mCi/mg.
Preferably, gelatinized starch in the gelatinized starch so- lution has a concentration of 1-10 wt%; the pharmaceutical starch suspension has an ionizing radiation intensity of 0.01-100 uCi/mL.
Preferably, the time interval of collecting urine for two times after the intragastric administration is 20 min to 2.5 h; the first urine collection time is 20 min to 2.5 h later after the intragastric administration.
Preferably, nitric acid in the mixed acid solution has a con- centration of 5.9-59 wt%, and hydrochloric acid has a concentra- tion of 3.3-33 wt.
Preferably, the soaking treatment time 1 s to 10 min.
Preferably, an equipment used for determining ionizing radia- tion counting of the “Hg is a y-ray counter connected with a so- dium iodide crystal scintillation detector having a large receiv- ing surface, a positron emission tomoscanner or a single photon emission computed tomoscanner.
The present invention provides a method for determining an absolute oral bicavailability of mercury in mercury sulfide or a mercury sulfide-containing material by a radioisotope ““’Hg, in- cluding following steps of: The present invention overcomes the interference technical barrier of mercury contamination generated during the process of sample collection, pre-treatment and deter- mination. Meanwhile, the present invention establishes a method for determining an absolute oral bioavailability (absorption rate) of mercury in mercury sulfide based on the amount of mercury re-
tention in vivo and the amount of mercury excreted via urine by labeling a sample to be tested with °%Hg (half-life is 46.612 days) via a highly sensitive y-ray counting method. The method will lay the foundation for the exposure risk assessment and toxi- cological and pharmacological studies of mercury in mercury sul- fide and mercury sulfide substances. Therefore, the present inven- tion is suitable for the fields such as, medicaments, environment and food for the determination of the absolute bioavailability of mercury in orally exposed mercury sulfide or mercury sulfide- containing substances. Compared with the prior art, the method provided by the present invention at least has the following ad- vantages:
In the present invention, a mixed acid solution is prepared by using nitric acid and hydrochloric acid which both have oxida- tion and chloride ion coordination abilities, and then the mixed acid solution is used to clean the sulfide or other forms of mer- cury which is derived from feces and remains on the body surface of the experimental animal. The present invention can solve the technical problem of hardly cleaning the residual mercury on the body surface of the experimental animal by other conventional cleaning methods, thereby ensuring the accurate determination of the absolute oral bioavailability of mercury in insoluble mercury sulfide.
In this present invention, ionizing radiation counting is utilized to calculate the absolute oral bicavailability of mercury in mercury sulfide or a mercury sulfide-containing substance. Fur- ther, the present invention has the advantages of reliability, high sensitivity; and the test value is absence of interference from stable mercury isotopes in the sample background.
Further, ““’HgS is prepared by a circuitous method in the pre- sent invention, which can effectively avoid the interference of radionuclides other than “Hg, such as *°’Hg, ’s and “0 introduced by a conventional direct preparation method on the determination of the absolute oral bicavailability of mercury.
Further, the present invention can directly determine the ionizing radiation counting (cpm) of *%Hg in the overall target sample (a syringe containing the pharmaceutical starch suspension or gelatinized starch solution before intragastric administration, a syringe containing the residual pharmaceutical starch suspension or gelatinized starch solution after intragastric administration, remaining bodies and urine of the experimental animals) using a y- ray counter connected with a sodium iodide crystal scintillation detector having a large receiving surface, a positron emission tomoscanner or a single photon emission computed tomoscanner.
Therefore, the present invention is free of complex pretreatments such as, digestion, dilution or homogenization (homogeneity) on the samples by inductively coupled plasma-mass spectrometry (ICP-
MS), inductively coupled plasma-optical emission spectrometer {ICP-0OES), atomic fluorescence spectrometry (AFS), atomic absorp- tion spectroscopy (ABS), colorimetry, direct mercury analyzer method based on the principle of pyrolysis, which effectively re- duces the interference on the test accuracy.
The present invention provides a method for determining an absolute oral bicavailability of mercury in mercury sulfide or a mercury sulfide-containing material by a radioisotope ““’Hg, in- cluding following steps of: mixing a gelatinized starch sclution with a sample to be tested to obtain a pharmaceutical starch suspension; the sample to be tested is mercury sulfide or a mercury sulfide-containing sub- stance which is labeled by “Hg; intragastrically administering the pharmaceutical starch sus- pension to experimental animals via a syringe as experimental group animals; intragastrically administering the gelatinized starch solution to experimental animals as blank control group an- imals; where before the intragastric administration, ionizing ra- diation counting of the total ***Hg in the pharmaceutical starch suspension or gelatinized starch solution of the syringe used for intragastric administration is determined; after the intragastric administration, ionizing radiation counting of the °%Hg in the re- sidual pharmaceutical starch suspension or gelatinized starch so- lution in the syringe was determined; collecting urine of the experimental group animals and blank control group animals after the intragastric administration re- spectively, then removing digestive tracts of the obtained experi- mental animals, and successively performing primary washing, im- mersion treatment with a mixed acid solution and secondary wash- ing, then collecting the remaining bodies of the obtained experi- mental animals; where the experimental animals were stimulated to urinate before intragastric administration and the obtained urine is discarded, and urine is collected continuously at a preset time interval after intragastric administration and before removing the digestive tracts; the mixed acid solution is a mixed solution of nitric acid and hydrochloric acid; respectively determining the collected urine and the remain- ing bodies of the experimental animals to obtain the ionizing ra- diation counting of *“*Hg in the urine and the ionizing radiation counting of *%Hg retained in the remaining bodies of the experi- mental animals, respectively; calculating the absolute oral bicavailability of mercury in the sample to be tested according to the formula shown in formula
I: absolute oral bioavailability of mercury (2) = (Nig—N.e)/ (Neo—
Nye) * 1003 Formula I; where, N,;-corrected zero-time ionizing radiation counting of the total °%Hg retained in urine and remaining bodies of the ex- perimental group animals;
Mo corrected zero-time ionizing radiation counting of the to- tal ““*Hg retained in urine and remaining bodies of the blank con- trol group animals;
N.;—corrected zero-time ionizing radiation counting of *°“Hg digested in the experimental group animals via intragastric admin- istration;
Ny—corrected zero-time ionizing radiation counting of ““’Hg digested in the blank control group animals via intragastric ad- ministration; the Ms; No, Ma and Ny are calculated according to the formu- las shown in Formula II, Formula III, Formula IV and Formula V, respectively:
Noo=Nag poay + Nao urine Formula II;
Noo=Noo toy + Moo urine Formula ITT;
Neo Nus before = Ney after Formula IV;
Nao-Nio before — Ngo after FOrmula V; where, Na toey corrected zero-time ionizing radiation counting of the *“Hg retained in the remaining bodies of the experimental group animals;
No urine Corrected zero-time ionizing radiation counting of the “Hg in urine of the experimental group animals;
Noe noey corrected zero-time ionizing radiation counting of the °%Hg retained in the remaining bodies of the blank control group animals;
Nig urine corrected zero-time ionizing radiation counting of the “®*Hg in urine of the blank control group animals;
Neo petore—COY¥rected zero-time ionizing radiation counting of the total “Hg in the pharmaceutical starch suspension in the sy- ringe used before intragastric administration to the experimental group animals;
Noo after corrected zero-time ionizing radiation counting of the “Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals;
Nas pefore Corrected zero-time ionizing radiation counting of the total °%Hg in the gelatinized starch solution in the syringe used before intragastric administration to the blank control group animals;
Nao after Corrected zero-time ionizing radiation counting of the “Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals; the Na pogys Nao arines Noo nodyr Nao urines Neo veforer Neo after; Nao before and Ns after are zero-time ionizing radiation counting obtained by respectively correcting ‘Hg ionizing radiation counting Nat bodys Nat urine? Ne boay r Not uriner Noy beforer Nat afterr Nat before and Nat after determined at t according to a “’Hg decay correction formula; the Nt pesy is ionizing radiation counting of the 2% Hg re- tained in the remaining bodies of the experimental group animals determined at t; and Nut wise is ionizing radiation counting of the
“Hg retained in urine of the experimental group animals deter- mined at t; the Nut voy is ionizing radiation counting of the “Hg re- tained in the remaining bodies of the blank control group animals determined at t; and Nx urine is ionizing radiation counting of the “Hg in urine of the blank control group animals determined at t; the Nu perere is ionizing radiation counting of the total “"’Hg in the pharmaceutical starch suspension in the syringe used before intragastric administration to the experimental group animals de- termined at t, and Nu after is ionizing radiation counting of the “Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals determined at t; the Nu perore is ionizing radiation counting of the total *““’Hg in the gelatinized starch solution in the syringe used before in- tragastric administration to the blank control group animals de- termined at t, and Ng arter is ionizing radiation counting of the “Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals determined at t; the ““Hg decay correction formula is shown in formula VI:
N; = Nye ™ Formula VI; where M-ionizing radiation counting of the °%Hg determined at Lt;
Ny-corrected zero-time ionizing radiation counting of the
A-decay constant, and the À=LIn(2)/t;2; ti,» is a half-life of the “Hg;
T-duration of time from the zero time as the starting point to the time point when the °%Hg ionizing radiation counting is de- termined, where the zero time refers to a certain time point prior to the intragastric administration of the first experimental ani- mal or the time point when the first experimental animal is admin- istered intragastrically; and the t20 s.
In the present invention, the absolute oral bioavailability (or absorption rate) refers to a ratio of the total amount of a certain substance which is taken orally and digested by the gas-
trointestinal tract into the body's circulation to the total amount of the substance taken orally.
In this present invention, a gelatinized starch solution is mixed with a sample to be tested to obtain a pharmaceutical starch suspension; the sample to be tested is mercury sulfide or a mercu- ry sulfide-containing substance which is labeled by “Hg. The pre- sent invention first describes a method for preparing °%Hg-labeled mercury sulfide (namely, “’HgS). In the present invention, the preparation method of the ““’HgS preferably includes following steps of: irradiating HgO with a neutron ray, and placing the irradiat- ed HgO such that the obtained ‘HgO decay weakens, thus obtaining 2 HgO; dissolving the ““’Hg0 into an inorganic acid, and mixing the obtained **%Hg% solution with a precipitant solution for reaction, then successively washing and drying the obtained precipitate to obtain the °%HgS; the precipitant in the precipitant solution is a soluble sulfide or thiosulfate.
In this present invention, HgO is irradiated with a neutron ray and then placed such that the obtained **’Hgo decay weakens, thus obtaining °°HgO. In the present invention, the HgO specifi- cally refers to non-radioactive HgO, unless otherwise specified.
In the present invention, the irradiation time is preferably 2 1 week, more preferably 2-4 weeks; the placing time is preferably 2 10 days, more preferably 10 days. In the examples of the present invention, the HgO is specifically placed into a double-layer quartz tube, sealed with an acetylene torch, irradiated under a neutron ray, and then placed in a lead box under protection. In the present invention, **'Hg0 is generated during the irradiation, and ‘*’Hg has a half-life of 64.14 h. :*'Hgo decay weakens during standing to obtain *%Hgo, and the “Hg has a half-life of 46.612 days.
After obtaining the °°Hgo, the *“*HgO is dissolved into an in- organic acid in this present invention, and the obtained ““Hg"* is mixed with the precipitant solution for reaction, and the obtained precipitate is successively washed and dried to obtain “**HgS. In the present invention, the inorganic acid is preferably hydrochlo-
ric acid or nitric acid; the inorganic acid preferably has a con- centration of 1-30 wt%, more preferably 3-10 wt%; specifically, ‘HgO may be dissolved with hydrochloric acid to obtain the ““HgCl; solution; and also, ““’HgO may be dissolved with nitric acid to ob- tain the °%Hg{(NO:)- solution. In the present invention, the precip- itant solution preferably has a concentration of 1 to 100 mg/mL, more preferably 10 to 50 mg/mL, further preferably 15 to 20 mg/mL; precipitant in the precipitant solution may be a soluble sulfide or thiosulfate. The soluble sulfide is preferably sodium sulfide or ammonia sulfide, and the thiosulfate is preferably sodium thio- sulfate; a molar ratio of the precipitant to ***Hg0 is preferably {1-2):1, more preferably (1.1-1.3):1. In the present invention, the reaction temperature is preferably from 70-180°C, more prefera- bly 100-120°C; the time is preferably 30 min to 24 hours, more preferably from 2 to 4 hours. During the reaction in the present invention, *“Hg*'forms a ““’HgS precipitate under the action of a precipitant, and the *’HgS precipitate is preferably collected by centrifugation or filtration. In the present invention, the wash- ing preferably includes hydrochloric acid washing and pure water washing successively. In the present invention, the hydrochloric acid washing is preferably to soak and wash the ““’HgS precipitate.
The hydrochloric acid used in the hydrochloric acid washing pref- erably has a concentration of 1-10 wt%, more preferably 3-5 wt$; the frequency of the hydrochloric acid washing is preferably 1-3 times, and the scaking time for each hydrochloric acid washing is preferably 1-5 min, more preferably 2-3 min. In the present inven- tion, the pure water washing is preferably to soak and wash the ‘VHgs precipitate after being washed by hydrochloric acid; the frequency of the pure water washing is preferably 3-5 times, and the soaking time in each pure water washing is preferably 1-5 min, more preferably 2-3 min. In the present invention, the °*%Hgs pre- cipitate is preferably collected by centrifugation or filtration after each washing. In the present invention, the drying is pref- erably freeze drying or oven drying; the specific operating condi- tions of the drying are not particularly limited in the present invention as long as sufficient drying is ensured. In the present invention, powdery “““HgS is obtained after drying.
In the present invention, the specific radioactivity of the ““*Hgs is preferably 1-20 mCi/mg, more preferably 5-15 mCi/mg, fur- ther preferably 7.5-10 mCi/mg. In this present invention, the spe- cific radioactivity of the °°HgS is specifically determined by a y-ray counter. *”HgS is prepared by a circuitous method in the present invention, which can effectively avoid the interference of radionuclides other than ‘Hg, such as ‘°’Hg, "S and "0 introduced by a conventional direct preparation method on the determination of the absolute oral bioavailability of mercury.
After obtaining the *%Hgs, the gelatinized starch solution is mixed with a sample to be tested in this present invention to ob- tain a pharmaceutical starch suspension; the sample to be tested is ‘%Hgs or *’HgS-containing substance. In the present invention, gelatinized starch in the gelatinized starch solution has a con- centration of preferably 1-10 wt%, more preferably 1-5 wt%. In the present invention, the method for preparing the gelatinized starch solution preferably includes following steps of: mixing starch with water to obtain a starch solution; heating the starch solu- tion to be boiling to obtain the gelatinized starch solution. In the present invention, the starch preferably includes Solanum tu- berosum starch or potato starch. The ‘“HgS-containing substance is not particularly limited in the present invention as long as a
PHgs-containing substance well known to a person skilled in the art may be used. In the examples of the present invention, °*’Hgs, an Anzhijinghua powder-“"’HgS mixture and an Ershiwuwei Zhenzhuwan pill-**Hgs mixture were specifically set as examples for descrip- tion. The Anzhijinghua powder-**’HgS mizture is obtained by mizing the Anzhijinghua powder with *”HgS by a mass ratio of 20.36:1, thereby simulating a Tibetan medicine with a mercury sulfide con- tent of 4.91% as an adjuvant. The Ershiwuwei Zhenzhuwan pill-***Hgs mixture is obtained by pulverizing the Ershiwuwei Zhenzhuwan pill and then mixing the obtained Ershiwuwei Zhenzhuwan pill powder with ““HgS by a mass ratio of 63.6:1, thereby simulating a com- pound Tibetan formulation in a proportion of 100 parts by weight of common compound formulation: 3 parts by weight of Tsothay (the
Tsothay contains 54% HgS}). In the present invention, the ionizing radiation intensity of the pharmaceutical starch suspension is preferably 0.01-100 uCi/mL, more preferably 0.1-50 uCi/mL, and further preferably 2.5-5 uCi/mL. In the present invention, since
HgS is a typical extremely insoluble compound and is easy to be precipitated due to its high density. The present invention pre- pares a pharmaceutical starch suspension based on a gelatinized starch solution, which is beneficial to uniformly dispersing the sample to be tested and avoids the problem of non-uniform concen- tration of the sample to be tested during intragastric administra- tion. Moreover, the gelatinized starch solution will not complex the trace amount of dissociated mercury ions, thus not affecting the experimental results. The sodium carboxymethyl cellulose solu- tion commonly used for suspension may complex the mercury ions, affecting the experimental results.
After obtaining the gelatinized starch solution and the phar- maceutical starch suspension, in this present invention, the phar- maceutical starch suspension is intragastrically administered to experimental animals via a syringe as experimental group animals; the gelatinized starch solution is intragastrically administered to experimental animals as blank control group animals; where be- fore the intragastric administration, ionizing radiation counting of the total “Hg in the pharmaceutical starch suspension or ge- latinized starch solution of the syringe used for intragastric ad- ministration is determined; after the intragastric administration, ionizing radiation counting of the total “**Hg in the residual pharmaceutical starch suspension or gelatinized starch solution in the syringe is determined. In the present invention, the experi- mental animal is preferably mice; in the examples of the present invention, healthy 8-12 week-old CD-1 mice or healthy 4 week-old
Wistar rats are used specifically. In the present invention, each group preferably includes 6-15 experimental animals when setting the groups of experimental animals. In the present invention, a ratio of the volume {intragastric volume} of the pharmaceutical starch suspension required by the experimental group animals to the body weight of the experimental animals is preferably {0.1- 0.3) mL:10 g. The operation mode of the intragastric administra- tion in the present invention is not particularly limited as long as a way well known to a person skilled in the art may be taken.
In the examples of the present invention, specifically, the phar- maceutical starch suspension is precisely administered intra- gastrically to the experimental animals with a 1 mL syringe. In the present invention, the volume of gelatinized starch solution required by the blank control group animals and the operation mode of intragastric administration are preferably the same as those in the experimental group animals, which will not be described in de- tail herein. In the present invention, at the end of intragastric administration, the intragastric volume and intragastric time of each experimental animal are preferably recorded. It should be noted that prior to the intragastric administration, the pharma- ceutical starch suspension of the present invention is preferably shaken well to avoid the problem of non-uniform concentration of the sample to be tested during the intragastric administration.
In the present invention, the experimental animals after be- ing administered intragastrically are preferably put to cages with corresponding numbers. The experimental animals preferably drink water and take food during the raising process. In the present in- vention, the experimental group animals and the blank control group animals are preferably raised in the light for 12 h and in the dark for 12 h; the raising temperature is preferably 18-28°C, more preferably 22°C; the humidity is preferably 30-70%, more pref- erably 40%.
Before intragastric administration, ionizing radiation count- ing of the total ***Hg in the pharmaceutical starch suspension or gelatinized starch solution of the syringe used for intragastric administration is determined; after the intragastric administra- tion, ionizing radiation counting of the ‘Hg in the residual pharmaceutical starch suspension or gelatinized starch solution in the syringe is determined. In the present invention, an equipment used for determining ionizing radiation counting of the “Hg is a y-ray counter connected with a sodium iodide crystal scintillation detector having a large receiving surface (e.g., Packard Auto- gamma Model 5530 or the like), a positron emission tomoscanner (PET) or a single photon emission computed tomoscanner (SPECT).
Further, the present invention can directly determine the ionizing radiation counting of “Hg in the overall target sample (a syringe containing the pharmaceutical starch suspension or gelatinized starch solution before intragastric administration, and a syringe containing the residual pharmaceutical starch suspension or gelat- inized starch solution after intragastric administration) prefera- bly using the above equipment. Therefore, the present invention is free of complex pretreatments such as, digestion, dilution or ho- mogenization (homogeneity) on samples by inductively coupled plas- ma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometer (ICP-0ES), atomic fluorescence spectrometry (AFS), atomic absorption spectroscopy (ABS), colorimetry, direct mercury analyzer method based on the principle of pyrolysis, which effectively reduces the interference on the test accuracy.
Urine of the experimental group animals and blank control group animals after being administered intragastrically is col- lected respectively, then digestive tracts of the obtained experi- mental animals are removed, and successively subjected to primary washing, immersion treatment with a mixed acid solution and sec- ondary washing, thus collecting the remaining bodies of the ob- tained experimental animals. In this present invention, the exper- imental animals are stimulated to urinate before intragastric ad- ministration and the obtained urine is discarded. Urine is contin- uously collected at a preset time interval after intragastric ad- ministration and before the removal of the digestive tracts. In this manner, the present invention may collect almost all the urine to minimize the loss of urinary mercury. The manner for stimulating urination is not specifically defined in this present invention as long as a manner well known to a person skilled in the art is used. Specifically, for example, the bladder of an ex- perimental animal is pressed to stimulate urination. The way of pressing the bladder of an experimental animal is preferably used to stimulate urination each time urine is collected in the present invention. In the present invention, the time interval of collect- ing urine for two times after the intragastric administration is 20 min to 2.5 h, more preferably, 1 h to 1.5 h; the first urine collection time is 20 min to 2.5 h later after the intragastric administration, more preferably, 1 h to 1.5 h. Urine is preferably collected in a disposable clean container and frozen or refriger-
ated at -20°C to 4°C in this present invention.
In the present invention, the time for removing the digestive tracts of the experimental animals is preferably 24 h to 240 h, more preferably 24 h to 48 h after intragastric administration. In this present invention, the experimental animals are anesthetized or euthanized preferably to achieve the removal of digestive tracts; the anaesthetic used for anaesthesia preferably includes chloral hydrate, pentobarbital sodium or urethane; the agent for euthanasia is preferably carbon dioxide. After anesthetizing or euthanizing the experimental animals, blood of the experimental animals is coagulated 30-60 min later preferably in the present invention, then the abdominal cavity and thoracic cavity are dis- sected to remove the digestive tract from esophagus to anus, then the obtained experimental animals are weighed and placed into a clean valve bag, and then frozen or refrigerated at -20°C to 4°C for further use. The advantages of the present invention in this way are as follows: on the one hand, blood of experimental animals is coagulated in vivo for overall test without blood collection to reduce the number of samples, on the other hand, the interference of the contents of the digestive tract on the determination of ““*Hgs may be eliminated.
After the digestive tract is removed, the present invention successively performs primary washing, scaking treatment with a mixed acid solution and secondary washing on the obtained experi- mental animals to collect the remaining bodies of the obtained ex- perimental animals. The mixed acid solution is a mixed solution of nitric acid and hydrochleric acid. In the present invention, ni- tric acid in the mixed acid solution preferably has a concentra- tion of 5.9-59 wt%, more preferably 20-30 wt%; hydrochloric acid preferably has a concentration of 3.3-33 wt%, more preferably 6-10 wt%. In the present invention, a mixed acid solution is prepared by using nitric acid and hydrochloric acid which both have oxida- tion and chloride ion coordination abilities, and then the mixed acid solution is used to clean the sulfide or other forms of mer- cury which is derived from feces and remains on the body surface of the experimental animal. The present invention can solve the technical problem of hardly cleaning the residual mercury on the body surface of the experimental animal by other conventional cleaning methods, thereby ensuring the accurate determination of the absolute oral bioavailability of mercury in insoluble mercury sulfide. In the present invention, the primary washing is prefera- bly to wash the experimental animals whose digestive tracts are removed with a detergent and then to wash with running water for 3-7 times; the detergent preferably includes a cleanser essence, a laundry detergent or a soap. In the present invention, the soaking treatment time with the mixed solution is preferably 1 s to 10 min, and more preferably for 0.5-2 min. In the present invention, the secondary washing is preferably to wash for 3 to 7 times with running water.
After collecting the urine and the remaining bodies of the experimental animals, the present invention determines the urine and the remaining bodies of the experimental animals, respectively to obtain the ionizing radiation counting of the total ““Hg in the urine and the ionizing radiation counting of the total ““*Hg in the remaining bodies of the experimental animals, respectively. In the present invention, the equipment for determining the urine and the remaining bodies of the experimental animals is preferably identi- cal to the above equipment for determining the ionizing radiation counting of the *““’Hg described above, which will not be described in detail herein. Specifically, in the present invention, the cul- ture dish for collecting urine is preferably subjected to overall determination to obtain ionizing radiation counting of the total “Hg which is absorbed by the body and excreted via the urine way; the remaining bodies of the experimental animals are subjected to overall determination to obtain ionizing radiation counting of the total *“Hg retained in the remaining bodies of the experimental animals.
In the present invention, the absolute oral bicavailability of mercury in the sample to be tested is calculated according to the formula shown in Formula I: absolute oral bicavailability of mercury (%) = (Ny—Nuy)/ (Neg—
Ng) x 100% Formula I; where, MN; corrected zero-time ionizing radiation counting of the total “**Hg retained in urine and remaining bodies of the ex-
perimental group animals;
Mo corrected zero-time ionizing radiation counting of the to- tal ““*Hg retained in urine and remaining bodies of the blank con- trol group animals;
No-corrected zero-time ionizing radiation counting of ““’Hg digested in the experimental group animals via intragastric admin- istration;
Ng-corrected zero-time ionizing radiation counting of “Hg digested in the blank control group animals via intragastric ad- ministration; the Ms, No; No and Na are calculated according to the formu- las shown in Formula II, Formula III, Formula IV and Formula V, respectively:
Nio= Nas poay + Nao urine Formula II;
N07Abs zoay + Mo urine Formula IIT;
Neo=Nog before = Neg atter Formula IV;
Nao-Nao verore = Nag atrer Formula V; where, Nao voay corrected zero-time ionizing radiation counting of the “Hg retained in the remaining bodies of the experimental group animals;
Nu urine— Corrected zero-time ionizing radiation counting of the “Hg in urine of the experimental group animals;
No poay—Corrected zero-time ionizing radiation counting of the 2%Hg retained in the remaining bodies of the blank control group animals;
No urine Corrected zero-time ionizing radiation counting of the “Hg in urine of the blank control group animals;
Neg before Corrected zero-time ionizing radiation counting of the total °°Hg in the pharmaceutical starch suspension in the sy- ringe used before intragastric administration to the experimental group animals;
Ng after corrected zero-time ionizing radiation counting of the “Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals;
Nao vetore Corrected zero-time ionizing radiation counting of the total “**Hg in the gelatinized starch solution in the syringe used before intragastric administration to the blank control group animals;
Nao after corrected zero-time ionizing radiation counting of the “Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals; the No posvr Nas uriner Noo nodyr Moo uciner Noo petorer Neo afters Nao before and Ns srter are zero-time ionizing radiation counting obtained by respectively correcting ““’Hg ionizing radiation counting Nat bogys Nat vrines Mot podys Not urines Net veforer Net afters Nat before and Na afer determined at t according to a ““*Hg decay correction formula; the Nit poay is ionizing radiation counting of the Hg re- tained in the remaining bodies of the experimental group animals determined at t; and N, wise is ionizing radiation counting of the ““*Hg retained in urine of the experimental group animals deter- mined at t; the Mt poy is ionizing radiation counting of the “Hg re- tained in the remaining bodies of the blank control group animals determined at t; and Nx urine is ionizing radiation counting of the *°Hg in urine of the blank control group animals determined at t; the Ne perore is ionizing radiation counting of the total Hg in the pharmaceutical starch suspension in the syringe used before intragastric administration to the experimental group animals de- termined at t, and Na after 18 ionizing radiation counting of the *Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals determined at t; the Na perore 1s ionizing radiation counting of the total 2%Hg in the gelatinized starch solution in the syringe used before in- tragastric administration to the blank control group animals de- termined at t, and Ni sree. is ionizing radiation counting of the °%Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals determined at t; the *%Hg decay correction formula is shown in formula VI:
Ny = Noe? Formula VI; where M-ionizing radiation counting of the “Hg determined at t;
N-corrected zero-time ionizing radiation counting of the ‘Hg;
A- decay constant, and the A=In(2)/t,:, tis is a half-life of the ‘Hg; t-duration of time from the zero time as the starting point to the time point when the *%Hg ionizing radiation counting is de- termined, where the zero time refers to a certain time point prior to the intragastric administration of the first experimental ani- mal or the time point when the first experimental animal is admin- istered intragastrically; and the t20 s.
In the present invention, the t is specifically defined as duration of time from the zero time as the starting point to the time point when the *%Hg ionizing radiation counting is deter- mined, where the zero time is taken as a certain time point of the period after preparing ““’HgS and before the intragastric admin- istration to the first experimental animal, or the zero time is taken as a time point when the first experimental animal is admin- istered intragastrically. The target sample specifically includes a syringe containing a pharmaceutical starch suspension or gelati- nized starch solution before intragastric administration, a sy- ringe containing a residual pharmaceutical starch suspension or gelatinized starch solution after intragastric administration, the remaining bodies and urine of the experimental animals. Specifi- cally, in the present invention, each corresponding time t is de- termined directed to the determination time of each target sample, and then, the ionizing radiation counting (M) of ‘>Hg determined at t is corrected by the formula as shown in Formula VI to obtain the corrected zero-time ionizing radiation counting (N,) of *“’Hg, and then Ny, Ay, Na and Ny are calculated according to the formu- las shown in Formula II, Formula III, Formula IV and Formula V, thus finally calculating the absolute oral bioavailability of mer- cury in the sample to be tested according to the formula shown in
Formula I. In this present invention, ionizing radiation counting is utilized to calculate the absolute oral bioavailability of mer- cury in mercury sulfide or a mercury sulfide-containing substance, which has the advantages of reliable data, high sensitivity; and the test value is absence of interference from stable mercury iso- topes in the sample background.
The technical solution of the present invention will be de- scribed more clearly and completely hereinafter with reference to the examples of the present invention. Apparently, the examples described are merely partial examples of the present invention in- stead of all the examples of the present invention. Based on the examples of the present invention, all the other examples obtained by a person skilled in the art without inventive effort shall fall within the protection scope of the present invention.
Example 1 (1) Preparation of other radionuclides-free °°HgS in a circu- itous manner 0.2 g HgO (0.9238 mmol) was placed into a double-layer quartz tube, sealed with an acetylene torch, and irradiated for 4 weeks under a neutron ray, and then placed into a lead box for 10 days under protection state to weaken the decay of the newly formed ‘go (**'Hg half-life was 64.14 h), thus obtaining *“*Hgo (***Hg half-life was 46.612 days), and then 10 mL hydrochloric acid with a concentration of 3 wt% (Suzhou Jingrui Chemical Co. Ltd., UP pu- rity) was added to dissolve the *°Hg0O to obtain a °%Hgcl: solution (the concentration of HgCl:was 25.0860 mg/mL, namely, 0.09238 mmol/mL); then 10 mL aqueous solution of sodium thiosulfate (Tian- jin Hengxing Chemical Reagent Factory, AR) (the concentration of sodium thiosulfate was 17.52744 mg/mL, namely, 0.1109 mmol/mL) was added. The obtained mixed solution was placed into a reaction ket- tle for reaction at 120°C for 4 h to generate *%Hgs precipitate (the molar ratio of sodium thiosulfate to HgCl, was 1.2: 1); at the end of the reaction, the *’HgS precipitate was collected by fil- tration, soaked and washed with diluted hydrochloric acid having a concentration of 5 wt% for 3 times, each scaking time was 3 min, and then soaked and washed with pure water for 5 times, each scak- ing time was 3 min; the “°*HgS precipitate was collected by filtra- tion, and subjected to vacuum freezing and drying ((FD-1D-50, Bei- jing BIOCOOL Experimental Instrument Co. Ltd.) to obtain °%Hgs powder, and the specific radioactivity of the ““’Hg was determined to be 7.5 mCi/mg by using a y-ray counter.
(2) Preparation of °°Hgs-starch suspension for intragastric administration
Potato starch (Qingdao Wanjiaxiang Food Co. Ltd., food grade) was put to a beaker, then pure water was added to the beaker ac- cording to the mass ratio of potato starch to pure water of 2:100, stirred and heated until boiling to obtain a gelatinized starch solution; after being cooled, the gelatinized starch solution was mixed with °°HgS powder to obtain a *“*HgS-starch suspension having an ionizing radiation intensity concentration of 5 uCi/mL and stored at room temperature. (3) Configuration of mice groups and treatments
Healthy 6-8 week-old CD-1 female mice were divided into an experimental group and a blank control group, 10 mice in each group. Experimental group mice: a ratio of the intragastric volume to the mice body weight was 0.1 mL:10 g; the ““’HgS-starch suspen- sion was accurately administered intragastrically using a 1 mL sy- ringe; blank control group mice: a ratio of the intragastric vol- ume to the mice body weight was 0.1 mL:10 g. The gelatinized starch solution was accurately administered by intragastrically using a 1 mL syringe. The intragastrical volume and intragastrical time of each mouse were recorded. It is noted that the ““*Hgs- starch suspension was shaken well before intragastric administra- tion to each animal, thus eliminating the problem of uneven drug concentration caused by intragastric administration. After the in- tragastric administration, the mice were placed into cages with corresponding number, drunk water and took food freely, and raised in the light for 12 h and in the dark for 12 h at a temperature of 22°C and a humidity of 40%. (4) Sample collection a) Urine collection. Before intragastric administration, the bladder of each mouse was pressed to stimulate urination, and the urine was discarded; the bladder was pressed to collect urine in a disposable clean petri dish every other 1 h after intragastric ad- ministration. Urine was collected continuously until euthanasia.
The collected urine was refrigerated at 4°C for further use. b} The remaining bodies of the mice were collected. 24 h lat- er after the administration, each mouse was euthanized with carbon dioxide, after the mouse died, blood in the mouse was coagulated after 30 min, and then the abdominal cavity and thoracic cavity were dissected to carefully remove the digestive tract from esoph- agus to anus, then the obtained mouse was weighed and placed into a clean valve bag, and then refrigerated at 4°C for further use.
Nitric acid (Suzhou Jingrui Chemical Co. Ltd., UP pure) and hydro- chloric acid (Suzhou Jingrui Chemical Co. Ltd., UP pure) were mixed in a glass container to obtain a mixed acid solution. Nitric acid in the mixed acid solution had a concentration of 20 wt% and the hydrochloric acid therein had a concentration of 10 wt%. The body of the mouse whose digestive tract was removed was washed with water containing detergent, then rinsed with running water for 5 times, blotted up with absorbent paper, and then soaked into the mixed acid solution. The soaking time was 30 s by time keeping of a stopwatch, and then the soaked mouse was washed with running water for 7 times, and remaining body of the cleaned test mouse was collected in a clean container for further use. (5) Direct determination on ionizing radiation counting (cpm) of ***Hg in the overall target sample
A y-ray counter (Packard Auto-gamma Model 5530, Packard In- strument Company, USA) connected with a sodium iodide crystal scintillation detector (7.6 cm diameter x 8.3 cm height) with a large test surface was used to directly determine the ionizing ra- diation counting of °%Hg in the overall target sample. The specif- ic operation was as follows: a) Before intragastric administration to each mouse, a y-ray counter connected with a sodium iodide crystal scintillation de- tector having a large detection surface was used to determine the ionizing radiation counting of the total “Hg in the pharmaceuti- cal starch suspension or gelatinized starch solution in the sy- ringe (the experimental group mouse corresponded to Na peforer and the blank control group mouse corresponded to Na perere) - After in- tragastric administration to each mouse, the radiation ionization counting of **Hg in the residual pharmaceutical starch suspension or gelatinized starch solution in the syringe (the experimental group mice corresponded to Nu afer; and the blank control group mice corresponded to Nx ater) was determined.
b) Urine of each mouse in the culture dish collected with urine was subjected to overall determination by a y-ray counter connected with a sodium iodide crystal scintillation detector hav- ing a large detection surface, thus obtaining the ionizing radia- tion counting of “Hg absorbed by the body and excreted through the urine (the experimental group mice corresponded to Ni urine, and the blank control group mice corresponded to Nt urine): c) The remaining body of the mouse was put to a y-ray counter connected with a sodium iodide crystal scintillation detector hav- ing a large detection surface for overall determination, so as to obtain the ionizing radiation counting of °%’Hg retained in the re- maining body of the mouse (the experimental group mice correspond- ed to Nt nosy, and the blank control group mice corresponded to Me body) + (6) Calculation on the absolute bioavailability (absorption rate) of mercury in oral HgS of mice
Based on the determined Na bodys Nav uriner Nor poeyr Not uriner Net be- forer Net atrverr Nit perore and Na after as well as the time t corresponding to the determination of each target sample, the °%Hg decay correc- tion formula (Formula VI) was used to respectively obtain Nas noeyy
Nag uriner Moo bedyr Noo uriner Neo beforer Neo afters Mio before and Nye after by cal- culation, and then N,;, Ns, Ms and Ni were calculated according to the formulas shown in Formula II, Formula III, Formula IV and For- mula V, and finally the absolute oral bicavailability of mercury in the sample to be tested was calculated according to the formula shown in Formula I, specifically as follows: absolute oral bioavailability of mercury (%) = (Ni—Nug)/ (Neo—
Ngg) x 100% Formula I; where, N,;-corrected zero-time ionizing radiation counting of the total *“ Hg retained in urine and remaining bodies of the ex- perimental group animals;
Mo corrected zero-time ionizing radiation counting of the to- tal ““*Hg retained in urine and remaining bodies of the blank con- trol group animals;
No-corrected zero-time ionizing radiation counting of ““’Hg digested in the experimental group animals via intragastric admin- istration;
Ny—corrected zero-time ionizing radiation counting of ““’Hg digested in the blank control group animals via intragastric ad- ministration; the No, No; Ns and Ni are calculated according to the formu- las shown in Formula II, Formula III, Formula IV and Formula V, respectively:
N‚o7Nag pody + Nag urine Formula IT;
Noo=Noo body + Mo urine Formula IIT;
Neg=Noo pesore = Nog after Formula IV;
Nag=Nag before — Ngo after Formula V; where, Nag roay corrected zero-time ionizing radiation counting of the “Hg retained in the remaining bodies of the experimental group animals;
No urine Corrected zero-time ionizing radiation counting of the ““*Hg in urine of the experimental group animals;
Noo poay—Corrected zero-time ionizing radiation counting of the “Hg retained in the remaining bodies of the blank control group animals;
Nog yrine— Corrected zero-time ionizing radiation counting of the “®"Hg in urine of the blank control group animals;
Neg perore—COrrected zero-time ionizing radiation counting of the total “®*Hg in the pharmaceutical starch suspension in the sy- ringe used before intragastric administration to the experimental group animals;
Nue after corrected zero-time ionizing radiation counting of the “Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals;
Nig vetore Corrected zero-time ionizing radiation counting of the total °%Hg in the gelatinized starch solution in the syringe used before intragastric administration to the blank control group animals;
Nao after corrected zero-time ionizing radiation counting of the “Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals; the No posyr Nao uriner Noo hodyr Moo ueiner Neo petorer Neo afters Na before and Ns after are zero-time ionizing radiation counting obtained by respectively correcting Hg ionizing radiation counting Nt pogy; Na urines Nt nodys Not urines Net pefores Net afters Not betore and Na afrer determined at t according to a “’Hg decay correction formula; the Nt pesy is ionizing radiation counting of the 2% Hg re- tained in the remaining bodies of the experimental group animals determined at t; and Nut uwiae is ionizing radiation counting of the “Hg retained in urine of the experimental group animals deter- mined at t; the Nut voay is ionizing radiation counting of the “Hg re- tained in the remaining bodies of the blank control group animals determined at t; and A+ urine is ionizing radiation counting of the 2%Hg in urine of the blank control group animals determined at t; the Nu verore is ionizing radiation counting of the total “Hg in the pharmaceutical starch suspension in the syringe used before intragastric administration to the experimental group animals de- termined at t, and Na after 18 ionizing radiation counting of the “Hg in the residual pharmaceutical starch suspension in the sy- ringe used after intragastric administration to the experimental group animals determined at t; the Ni perore is ionizing radiation counting of the total Hg in the gelatinized starch solution in the syringe used before in- tragastric administration to the blank control group animals de- termined at t, and Ng arter 18 ionizing radiation counting of the *Hg in the residual gelatinized starch solution in the syringe used after intragastric administration to the blank control group animals determined at t; the ‘Hg decay correction formula is shown in formula VI:
Ny = Nge™ Formula VI; where M-ionizing radiation counting of the “Hg determined at Lt;
N‚-corrected zero-time ionizing radiation counting of the 2g;
A-decay constant, and the A=In{(2)/t 2, ti: is a half-life of the ‘Hg; t-duration of time from the zero time as the starting point to the time point when the °%Hg ionizing radiation counting is de-
termined, where the zero time refers to a certain time point prior to the intragastric administration of the first experimental ani- mal; and the t20 s.
Finally, according to Formula I, the absclute bicavailability of mercury in oral HgS of mice was calculated to be 0.0127 + 0.0022% (Mean + SD), namely, 0.000127.
Example 2
Example 2 was operated according to the method of Example 1 and only differed from Example 1 in that:
In step (1), sodium sulfide was reacted with the °%pgcl. to generate ““HgS precipitate, specifically as follows, 10 mL aqueous solution of sodium sulfide (the sodium sulfide concentration was 8.6552 mg/mL, namely, 0.1109 mmol/mL) was added to 10 mL “®*HgCl, solution (the HgCl, concentration was 25.0860 mg/mL, namely, 0.09238 mmol/mL), and mixed well for reaction at room temperature for 30 min to generate *“*HgS precipitate (the molar ratio of sodi- um sulfide to HgCl, was 1.2:1). At the end of the reaction, the ‘VHgs precipitate was filtered and collected, soaked and washed with diluted hydrochloric acid having a concentration of 5 wt% for 3 times; each soaking time was 3 min, and then the mice were soaked and washed with pure water for 5 times, each soaking time was 3 min; the *“"HgS precipitate was filtered and collected, and vacuum frozen-dried (FD-1D-50, Beijing BIOCOOL Experimental In- strument Co., Ltd.) to obtain “**HgS powder, and the specific radi- oactivity of “Hg was determined to be 7.7 mCi/mg by a y-ray coun- ter.
In step (2), a gelatinized starch solution was used to pre- pare an Anzhijinghua powder-*"*HgS-starch suspension having an ion- izing radiation intensity concentration of 2.5 pCi/mL, specifical- ly as follows: the Anzhijinghua powder (an internal formulation of
Qinghai Provincial Tibetan Hospital) and “°’HgS powder were pre- pared into 1 g Anzhijinghua powder-“*HgS mixture by a mass ratio of 20.36:1, so as to simulate a Tibetan medicine with a mercury sulfide content of 4.91% as an adjuvant (when the adjuvant = 10 parts by weight of Anzhijinghua powder + 1 part by weight of Tso- thay, the Tsothay contained 54% HgS). The Anzhijinghua powder- ““Hgs mixture was then mixed with the gelatinized starch solution to prepare an Anzhijinghua powder-‘"’HgsS-starch suspension with an ionizing radiation intensity concentration of 2.5 uCi/mL (namely, in this example, the “”HgS-starch suspension in Example 1 was re- placed with the Anzhijinghua powder-“’HgsS-starch suspension subse- quently).
Nitric acid in the mixed acid solution of step (4) had a con- centration of 30 wt%, and the hydrochloric acid therein had a con- centration of 6 wt.
In step (6), the absolute bioavailability (absorption rate) of mercury in the Anzhijinghua powder-°"’HgS mixture orally taken by the mice was calculated according to the formula shown in For- mula I, being 0.0289 + 0.0019% (Mean + SD), namely, 0.000289.
Example 3
Example 3 was operated according to the method of Example 1 and only differed from Example 1 in that:
In step (2), a gelatinized starch solution was used to pre- pare an Ershiwuwei Zhenzhuwan pill-*"*HgS-starch suspension having an ionizing radiation intensity concentration of 1 uCi/mL, specif- ically as follows: the Ershiwuwei Zhenzhuwan pill (Tibet Ganlu Ti- betan Medicine Co. Ltd., CFDA (Approval) 754020080) was pulverized with a micro-pulverizer (type BJ-150); the obtained powdered Er- shiwuwei Zhenzhuwan pill and **Hgs powder were prepared into 1 g
Ershiwuwei Zhenzhuwan pill-“’HgS mixture by a mass ratio of 63.6:1, so as to simulate a compound Tibetan formulation with a ratio of 100 parts by weight of common compound formulation: 3 parts by weight of Tsothay (the Tsothay contained 54% HgS). The
Ershiwuwei Zhenzhuwan pill-°°HgS mixture was then mixed with a ge- latinized starch solution to prepare an Ershiwuwei Zhenzhuwan pill-“*Hgs-starch suspension with an ionizing radiation intensity concentration of 2.5 uCi/mL (namely, in this example, the ?%Hgs- starch suspension in Example 1 was replaced with the Ershiwuwei
Zhenzhuwan pill-*“HgS-starch suspension subsequently).
The experimental animals in step (3) were healthy 4-week-old female Wistar rats, and 7 rats in each group.
Nitric acid in the mixed acid solution of step (4) had a con- centration of 30 wt%, and the hydrochloric acid therein had a con- centration of 6 wt%.
In step (6), the absolute bicavailability (absorption rate) of mercury in the Ershiwuwei Zhenzhuwan pill-**Hgs mixture orally taken by the mice was calculated according to the formula shown in
Formula I, being 0.0183 + 0.0005% (Mean + SD), namely, 0.000183.
Test example
To compare the cleaning effect on the ““HgS interference de- rived from feces and adsorbed on the surface of experimental ani- mals by the washing method of a mixed acid prepared by nitric acid and hydrochloric acid with oxidation and coordination abilities with that by conventional washing methods, the following experi- ment was designed: (1) 8-12 week-age CD-1 male mice were divided into 3 groups: namely, ““’HgS-starch suspension smearing + mixed acid cleaning group, -“’HgS-starch suspension smearing + conventional cleaning group, blank + conventional cleaning group, 6 mice in each group.
Nitric acid in the mixed acid liquid had a concentration of 30 wt3, and hydrochloric acid herein had a concentration of 6 wt. (2) Mice in the *%HgS-starch suspension smearing + mixed acid cleaning group and “®’HgS-starch suspension smearing + conventional cleaning group were smeared with ““°HgS suspension with an ionizing radiation intensity of 1 pCi/mL, while the mice in the blank + conventional cleaning group was not treated like this. (3) The mice were euthanized with carbon dioxide and the di- gestive tract was removed to obtain the remaining bodies of the mice without digestive tract. (4) The remaining bodies of the above 6 mice in the *’Hgs- starch suspension smearing + mixed acid cleaning group smeared with the above ““HgS-starch suspension were washed with water con- taining detergent, then washed with running tap water for 5 times, blotted up with absorbent paper, then soaked in the mixed acid for 30 s, and then washed with running tap water for 7 times. The above 6 mice in the *%Hgs-starch suspension smearing + convention- al cleaning group smeared with the above “’Hgs-starch suspension were washed with water containing detergent, then washed with run- ning tap water for 5 times, and then washed with running tap water for 7 times continuously (namely, the step of soaking into the mixed acid solution was omitted); 6 mice in the blank control group were washed with water containing detergent, then washed with running tap water for 5 times, and then continuously washed with running tap water for 7 times, and placed into a clean con- tainer for future use. (5) A y-ray counter (Packard Auto-gamma Model 5530, Packard
Instrument Company, USA) connected with a sodium iodide crystal scintillation detector (7.6 cm diameter x 8.3 cm height) having a large test surface was used to determine the ionizing radiation counting of the “Hg in the remaining bodies of the above each group of mice. The specific results are shown in Table 1.
Table 1 Clearing result of the “"’Hgs interference derived from faeces absorbed on the body surface of mice (n = 6, MeantSD) lonizing radiation counting of the
Experimental group | total “Hg in the remaining bodies | Mean, cpm RSD, % of the mice, cpm
Blank + conven- tional cleaning 505.50 +21.87 4.32770476 group pension smearing+ 9651.50 £3736.68*** | 38.71604548 conventional clean- pension smearing + rived acid cleaning 547.83 £26.11 4.766304828
Note: compared with the blank + conventional cleaning group, "xxx"! means p <0.001.
The above results show that a mixed acid solution is prepared by using nitric acid and hydrochloric acid which both have oxida- tion and chloride ion coordination abilities, and then the mixed acid solution is used to clean the ““’HgS derived from feces ad- sorbed on the body surface of the experimental animals. The pre- sent invention has significant effects, and is superior to the conventional washing method and can ensure the accuracy of deter- mination of the absolute oral bicavailability of mercury in mercu- ry sulfide substances.
What is described above are merely preferred embodiments of the present invention. It should be indicated that a person skilled in the art may make several improvements and embellish- ments within the principle of the present invention. Moreover, these improvements and embellishments shall be also regarded with- in the protection scope of the present invention.
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