High specific fluorescent method staining of zinc-insulin complex in pancreatic B-cells

Authors demonstrated results using of high specific fluorescent method histochemical staining of Zn+2-ions in pancreatic B-cells. It was showed in diabetic animals and in animals past mobilization of insulin from B-cells a simultaneous decreasing as of amount of insulin as of Zn+2-ions in cytoplasm of B-cells. Meanwhile for- mation in B-cells of chemical complexes of derivatives of Diethyldithiocarbamic acid with Zn+2-ions result negative fluorescent reaction for Zn+2-ions but positive reaction for insulin using of insulin staining methods.

It is known that pancreatic B-cells contained a large amount of ions of Zinc [1–3] as salivary glands and prostate. In B-cells zinc ions take part in processes of biosynthesis of insulin as in of storage of insulin by forming of zinc-insulin complex [4, 5]. Pancreas of rat, rabbit, dog, cat, some fish, human, birds, mice, ham- ster, porcine, hoerst, contained a large amount of zinc. Using of electron microscopy histochemical method it was showed that that zinc concentrated in B-cells in B-granules only contained deposited form of insulin [6] and destruction of B-cells caused by Dithizon which formed in B-cells toxic complexes with zinc-ions, start- ed by destruction of B-granules [7].

Widely known methods staining of insulin as immunohistochemical, immunofluorescent, diethyl- pseudoisocyanine and some other methods are specific for insulin only but not for staining of zinc ions. Very often in diabetes and intact B-cells there are is a quantity correlation between insulin and zinc ions content: decreasing of insulin content accompanied by decreasing of amount of zinc ions and in opposite, in intact B-cells a large amount of insulin accompanied by a large amount of zinc-ions. Meanwhile for estimate abil- ity of B-cells for storage of insulin in cells it is necessary to use method of staining of zinc-ions whereas staining of insulin is indirect method for to estimate concentration of zinc ins in B-cells.

Aim of work: to study result using of high specific fluorescent methods revealing of zinc ions by using of 8-para(toluenesulphonylamino)quinolin (TSQ), a high specific for Zn+2-ions reagent [8, 9] which formed complex «zinc-TSQ». TSQ is a derivative of 8-oxyquinolin and synthesis was elaborated by Prof. N.N.Voroshzov in 1930 [10]. In UV-light with maximum of absorbance as 360–370 nm, this complex fluo- resces brightly green light [11]. Specificity for zinc ions of this method was confirmed in vitro by interaction of pure zinc ions with TSQ that result intensive green fluorescence of solution; using of spectral analysis it was confirmed presence in solution of «zinc-TSQ» complex and correlation of maximum of absorbance of this complex with pure complex synthezed in vitro. Sensitivity of this method is high and concentration of zinc as 10–7–10–8 revealed using it [8]. This same time in TSQ is TSQ possess a high diabetogenic activity and injection of 40–50 mg/kg result necrosis, destruction and death of absolute majority of B-cells within 20–30 min past formation of complex «zinc-TSQ» in B-cells [12].

Materials and methods

8 rabbits (2450–2680 g) and 3 Guinea Pigs (320–370 g). Group 1: 3 animals with diabetes caused by Dithizon (48,8–51,6 mg/kg); Group 2: intact rabbits; Group 3: 3 animals past administration of Na salt of diethyldithiocarbamic acid (DCA) (504 and 987 mg/kg) that result temporary non toxic binding of zinc in B-cells; Group 4: 2 Guinea Pigs past administration of DZ (49,5–52,8 mg/kg) and 2 intact Guinea Pig.

Preparing of  Dithizon  water-ammonia  solution:  Dithizon  (Avocado  Chem.  Company,  USA)  — 200 mg.+ 15 ml. of bi-distilled water + 0,25 ml of 25 % ammonia, 10 min mixing on temperature +70º Cel- sius.

Staining technologies

Fluorescent reaction with 0,04 % ammonia solution of TSQ was used. Staining procedures: a few drops of TSQ solution place on frozen sections for 10 sec.; 3 times wash by distilled water and investi- gation on UV-light microscope with measuring of intensity of fluorescence (intensity of fluorescence in con- trol was accepted for 1,00; length of wave of light — 360 nanometers. For quantitative estimation of results of measuring intensity of fluorescence parameter K was calculated as relation: Intensity of fluorescence of B-cells IF1/ Intensity of fluorescence of exocrine tissue cells IF2 (IF1/IF2); intensity of staining of exocrine tissue cells was accepted for 1,00 using of histofluorimetrical complex constructed by G.G.Meyramov and coll. [13] (Fig. 1).

Figure 1. Histofluorimetrical complex for measurement amount of zinc-insulin complex in pancreatic B-cells 

For insulin staining the Immunohistochemical (anticorps for insulin from DAKO, Denmark) and Pseudoisocyanine  [14] (SERVA, Germany)  methods  was  used.  For quantitative estimation  of  results of measuring intensity of fluorescence parameter K was calculated as relation: Intensity of fluorescence of B-cells IL1/ Intensity of fluorescence of exocrine tissue cells IL2 (IL1/IL2); intensity of staining of exocrine tissue cells was accepted for 1,00. For quantitative estimation of results of measuring density of staining B-cells by Immunohistochemical method parameter K was calculated as relation: Density of staining of B-cells IG1/ Density of staining of exocrine tissue — IG2 (IG1/IG2); intensity of staining of exocrine tissue cells was accepted for 1,00.

For histological analysis Victoria-4 histochemical method (MERCK, Germany) was used [15–18].

Results

1. Group 1. Immediately past injection of Dithizon (DZ) negative reaction for zinc ions was revealed in B-cells contrary to positive reaction for insulin (fig.2.1, 2) that is determined by binding of all amount of zinc in cells by DZ. As result zinc ions not formed with TSQ visible fluorescent complex in cytoplasm of B-cells. Intensity of fluorescence of B-cells: K(IF1/IF2) = 1,04±0,02; control: intact B-cells: K = 2,02±0,07 (p < 0,001). Insulin content in B-cells: K(IG1/IG2) = 1,88±0,02 IL1/IL2 = 2,06±0,08 (Table).
2. Group 2. Animals with diabetes caused by injection of DZ (50,2 and 47,6 mg/kg) 7 days ago. Nega- tive reaction as for zinc-ions with TSQ as for insulin revealed in B-cells on frozen sections of pancreas tissue (fig. 2.3, 2.4) that demonstrated absence in cytoplasm of B-cells as of zinc-ions as of insulin in result of ne- crosis and destruction of cells: K(IF1/IF2) = 1,08±0,03; control: intact B-cells: K = 2,00±0,08 (p < 0,001). Insulin content in B-cells: K(IG1/IG2) = 1,12±0,02; IL1/IL2 = 1,07±0,06 (Table).
3. Group Injection of Na salt of diethyldithiocarbamic acid (DCA) result forming in cytoplasm of B-cells of not toxic complex zinc-DCA for a few hours. A negative reaction for zinc-ions (fig. 2.5) was re- vealed in B-cells past injection of DCA that is explained by formation of complex zinc-DCA that is why TSQ not formed fluorescent complex zinc-TSQ in cells [7]: K = K(IF1/IF2) = 1,02±0,04; control: intact not diabetogenic temporary binding of zinc-ions  by  DCA;  UV-light microscopy;

Figure 2. Zinc-ions and insulin content in B-cells of intact and experimental rats 

Group B-cells of Guinea Pig contrary to many other animals (rabbit, rat, dog, fish, cat, hamster, porcine) not contained zinc ions and biochemical nature of processes of insulin storage in B-cells of Guinea Pig now not cleared yet.

Negative reaction for zinc-ions with TSQ was revealed in B-cells: K = (IF1/IF2) = 0,98±0,04;   control: intact B-cells of rat: K = 1,97±0,06 (p < 0,001). Insulin content in B-cells: IL1/IL2 = 1,91±0,05 (Table). 

Insulin and Zinc content in pancreatic B-cells (parameter K)

T a b l e

Discussion

In 1961 E.Boshevolnov and G.Serebrarakova informed about ability of TSQ, a derivative of 8-oxyquinolin, to form in vitro complexes with Zn+2-ions and with ions of Cadmium (Fig. 3, 4).

Figure 3. 8-para(toluene- sulphonylamino)quinolin (TSQ)

Figure 4. Complex Zn+2 – 8-para(toluene- sulphonylamino)quinolin (TSQ)

Zn+2-TSQ complex radiates intensive green fluorescence under UV-light 360–370 nm length of wave and Cd-TSQ — intensive yellow fluorescence that was confirmed by spectral analysis of spectrum of ab- sorbance. Past long time prolonging testing in Institute of High Pure Chemicals (Moscow) TSQ was pro- posed as fluorescent reagent for identification of very small amounts of zinc in solutions and tissues. Later in laboratory of Lasaris Y.A. and coll., Karaganda, TSQ was tested for revealing in vitro and in intact and dia- betic animals of a large amount of zinc-ions. TSQ is high specific reagent for staining of zinc-ions in pancre- atic B-cells. Now there are not other methods for revealing of zinc-ions in B-cells. It is known that zinc-ions take part in processes of storage of insulin by formation of complex zinc-insulin in B-cells. Very often there are parallelism between content of zinc and insulin in cytoplasm of B-cells and is possible to stain insulin in B-cells for estimate a content of zinc-ions in cells.

Results of using for many years of this method revealing of zinc-ions showed that in 3 cases method demonstrated a full coincidence with content of insulin in B-cells: 1) in intact animals; 2) in animals with experimental diabetes; 3) in animals after removing of zinc-insulin complex from B-cells by drugs. That is why this method can be used not only for estimate of zinc-ions content in B-cells but for insulin content too.

In one case results of TSQ-reaction can not correspond to quantity contained of zinc-ions in B-cells: some chemicals formed complexes with zinc in B-cells for short period and this time in fluorescent reaction for zinc will be negative despite of presence a large amount of metal in cytoplasm of cells.

This method demands following conditions: for fixing of tissue of a pancreas to use the alcohol sated with hydrogen sulfide (H2S) or to use sections of frozen pancreas tissue. Filters for UV-microscopy: UV-filter between UV-lamp and microscope and yellow filter for ocular of microscope.

Acknowlegement

Authors  are  thankful  for  methodical  assistance  and  financial  supporting  of  investigation  to  Prof. G.G.Meyramov. 

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