Journal of Solid Tumors 2016, Vol. 6, No. 1 BRIEF REPORT Studies on antitumor activity spectrum of doxycycline Bo Chen1, 3, Hong-gang Zhou2, 3, Wei Wang2, 3, Wen-guang Gu2, 3, Dong Zhao2, 3, Peng Wang∗3 1College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China 2State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, China3Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of Biomedicine, Tianjin, China
0542946food_a.fmBOEHRINGER MANNHEIM / R-BIOPHARM
Enzymatic BioAnalysis / Food Analysis
for the determination of urea and ammonia in foodstuffs and
other materials and for the determination of nitrogen after
For in vitro use only Store at 2-8° C
Kjeldahl-digestion (see pt. 12.2)
Cat. No. 10 542 946 035
Test-Combination for approx. 25 determinations each
For recommendations for methods and standardized procedures see ref. (2) Principle (Ref. 1)
If the absorbance A2 decreases constantly, extrapolate the absorbance to Urea is hydrolyzed to ammonia and carbon dioxide in the presence of the the time of the addition of solution 4 (GlDH).
enzyme urease (1).
Determine the absorbance differences (A1-A2) for both, blanks and samples.
Subtract the absorbance difference of the blank from the absorbance difference of the corresponding sample.
In the presence of glutamate dehydrogenase (GIDH) and reduced nicotinamide- A = (A1-A2)sample - (A1-A2)blank adenine dinucleotide (NADH), ammonia reacts with 2-oxoglutarate to This results in A L-glutamate, whereby NADH is oxidized (2).
urea + ammonia (from urea sample) and ammonia (from ammonia sample).
(2) 2-Oxoglutarate + NADH + NH + L-glutamate + NAD+ + H2O The difference of these values results in A urea.
The amount of NADH oxidized in the above reaction is stoichiometric to the The measured absorbance differences should, as a rule, be at least 0.100 amount of ammonia or with half the amount of urea, respectively. NADH is absorbance units to achieve sufficiently precise results (see "Instructions for determined by means of its light absorbance at 334, 340 or 365 nm.
performance of assay" and "Sensitivity and detection limit", pt. 4).
The Test-Combination contains
If the absorbance differences of the samples (Asample) are higher than 1. Bottle 1 with approx. 60 ml solution, consisting of: 1.000 (measured at 340 nm or Hg 334 nm respectively) or 0.500 (measured triethanolamine buffer, pH approx. 8.0; 2-oxoglutarate, approx. 220 mg at 365 nm), the concentration of urea (or ammonia) in the sample solution is 2. Bottle 2 with approx. 50 tablets; each tablet contains: too high. The sample is to be diluted according to the dilution table in that NADH, approx. 0.4 mg 3. Bottle 3 with approx. 0.7 ml urease solution, approx. 80 U 4. Bottle 4 with approx. 1.2 ml glutamate dehydrogenase solution, According to the general equation for calculating the concentration: Preparation of solutions
1. Use contents of bottle 1 undiluted.
2. Dissolve one tablet of bottle 2 with one ml solution of bottle 1 in a beaker
= final volume [ml] or in a reagent tube for each assay (blank and samples) depending onthe number of determinations. Use forceps for taking the tablets out of = sample volume [ml] bottle 2. This results in reaction mixture 2*.
MW = molecular weight of the substance to be assayed [g/mol] 3. Use contents of bottle 3 undiluted.
= light path [cm] 4. Use contents of bottle 4 undiluted.
= extinction coefficient of NADH at: Stability of reagents
340 nm = 6.3 [l × mmol-1 × cm-1] Solution 1 is stable at 2-8°C (see pack label).
Hg 365 nm = 3.4 [l × mmol-1 × cm-1] Bring solution 1 to 20-25°C before use.
Hg 334 nm = 6.18 [l × mmol-1 × cm-1] Tablets 2 are stable at 2-8°C (see pack label).
It follows for urea: Reaction mixture 2 is stable for 3 days at 2-8°C.
Bring reaction mixture 2 to 20-25°C before use.
× Aurea[g urea/l sample The contents of bottle 3 and 4 are stable at 2-8°C (see pack label).
× 1.00 × 0.100 × 2 × 1000 340 nm, Hg 365 nm or Hg 334 nm 1.00 cm light path × Aammonia[g ammonia/l × 1.00 × 0.100 × 1000 If the sample has been diluted on preparation, the result must be multiplied Read against air (without a cuvette in the light path) or against water by the dilution factor F.
0.3-14 g urea/assay3 or 0.2-8 g ammonia/assay3(in 0.100-2.000 ml sample volume) Pipette into cuvettes reaction mixture 2* sample solution** Mix***, and read absorbances of the solutions (A1) after approx. 5 min at The absorption maximum of NADH is at 340 nm. On spectrophotometers, measurements 20-25°C. Start reaction by addition of: are taken at the absorption maximum; if spectralline photometers equipped with a mercury
vapor lamp are used, measurements are taken at a wavelength of 365 nm or 334 nm.
If desired, disposable cuvettes may be used instead of glass cuvettes.
Mix***, wait for completion of the reaction (approx. 20 min) and read See instructions for performance of assay absorbances of the solutions (A2). For simplification of the assay performance it is also possible to pipette directly 1.000 ml of If the reaction has not stopped after 20 min, read absorbances in 2 min solution 1 into the cuvette and add 1 tablet from bottle 2. After dissolution of the tablet with intervals until the absorbances decrease constantly over 2 min.
the aid of a spatula continue working as described in the procedure. The difference involume of approx. 1% (increase of volume by 1 tablet per 3.040 ml assay volume) has to betaken into account in the calculation by multiplication of the result with 1.01.
Rinse the enzyme pipette or the pipette tip of the piston pipette with sample solution beforedispensing the sample solution.
*** For example, with a plastic spatula or by gentle swirling after closing the cuvette with Parafilm (trademark of the American Can Company, Greenwich, Ct., USA) 0711.11 748 530001 ➂
When analyzing solid and semi-solid samples which are weighed out for sample preparation, the result is to be calculated from the amount weighed: In a double determination using one sample solution, a difference of 0.005 to 0.015 absorbance units may occur. With a sample volume of v = 0.100 ml urea [g/l sample solution] and measurement at 340 nm, this corresponds to an urea concentration of weightsample in g/l sample solution approx. 0.7-2 mg/l. (If the sample is diluted during sample preparation, the result has to be multiplied by the dilution factor F. If the sample is weighed in for sample preparation, e.g. using 1 g sample/100 ml = 10 g/l, a difference of ammonia [g/l sample solution] 0.007- 0.02 g/100 g can be expected.) sample in g/l sample solution The following data for the determination of urea have been published in the Instructions for performance of assay
The amount of urea (ammonia) present in the assay has to be between 0.3 g and 14 g (0.2 g and 8 g). In order to get a sufficient absorbance difference, the sample solution is diluted to yield an urea (ammonia)concentration between 0.02 and 0.14 g/l (0.01 and 0.08 g/l).
Analysis of swimming-pool water: s(r) = ± 0.066 mg/l s(R) = ± 0.076 mg/l Estimated amount of urea (ammonia) s(r) = ± 0.044 mg/l s(R) = ± 0.067 mg/l < 0.14 g ( 0.08 g) (r) = ± 0.025 mg/l 0.14-1.4 g (0.08-0.8 g) (R) = ± 0.060 mg/l 1.4-14 g (0.8-8.0 g) Interference/sources of error
If the measured absorbance difference (A) is too low (e.g. 0.100), the During protein precipitation with perchloric acid which is to be carried out in sample solution should be prepared again (weigh out more sample or dilute foodstuffs, protein fragments are occasionally obtained. These protein less strongly) or the sample volume to be pipetted into the cuvette can be fragments are kept in solution and may gradually form ammonia in alkaline increased up to 2.000 ml. The volume of water added must then be reduced buffer systems leading to creep reactions. This formation of ammonia is very to obtain the same final volume in the assays for sample and blank. The new low and can be differentiated and calculated from the ammonia content of sample volume v must be taken into account in the calculation.
the sample by extrapolation of the absorbance A2 to the time of addition of solution 4 (GlDH).
The common ingredients of foodstuffs do not interfere with the assay of urea 2.1 Use only freshly distilled water for the assay.
and ammonia. Only high concentrations of tannins in fruit juices may causean inhibiton of the GIDH reaction. Fruit juices should therefore always be 2.2 Work in an atmosphere free from ammonia (ban smoking in the treated with PVPP.
As high concentrations of heavy metals cause turbidity, they make a reliabledetermination of ammonia difficult. In most cases high concentrations of Specificity (Ref. 1)
metal ions can be removed as hydroxides by alkalization of the sample solu- The method is specific for urea and ammonia.
tion (pH 7.5).
In the analysis of commercial urea and ammonium sulfate results of approx.
100% have to be expected.
Sodium thiosulfate, occasionally added to samples of swimming-pool water,does not interfere with the assay up to 1 mg per assay.
4. Sensitivity and detection limit (Ref. 1.4)
Recognizing interference during the assay procedure
The smallest differentiating absorbance for the procedure is 0.005 8.1 If the conversion of urea and ammonia has been completed according absorbance units. This corresponds to a maximum sample volume v = 2.000 ml to the time given under "Procedure", it can be concluded in general that and measurement at 340 of an ammonia concentration of 0.02 mg/l sample no interference has occurred.
solution, resp. of an urea concentration of 0.04 mg/l (if v = 0.100 ml, thiscorresponds to 0.4 mg ammonia/l, resp. 0.8 mg urea/l sample solution).
8.2 On completion of the reaction, the determination can be restarted by adding urea and/or ammonium chloride or ammonium sulfate (qualita- The detection limit of 0.08 mg ammonia/l, resp. 0.15 mg urea/l is derived tive or quantitative): if the absorbance is altered subsequent to the from the absorbance difference of 0.020 (as measured at 340 nm) and a addition of the standard material, this is also an indication that no inter- maximum sample volume v = 2.000 ml.
ference has occurred.
8.3 Operator error or interference of the determination through the presence of substances contained in the sample can be recognized by Linearity of the determination exists from approx. 0.2 g ammonia/ carrying out a double determination using two different sample assay (0.08 mg ammonia/l sample solution; sample volume v = volumes (e.g. 0.100 ml and 0.200 ml): the measured differences in 2.000 ml) to 8 g ammonia/assay (0.08 g ammonia/l sample solution; absorbance should be proportional to the sample volumes used.
sample volume v = 0.100 ml), resp. from 0.3 g urea/assay (0.15 mg urea/lsample solution; sample volume v = 2.000 ml) to 14 g urea/ assay (0.14 g When analyzing solid samples, it is recommended that different urea/l sample solution; sample volume v = 0.100 ml).
quantities (e.g. 1 g and 2 g) be weighed into 100 ml volumetric flasks.
The absorbance differences measured and the weights of sample used 6. Precision
should be proportional for identical sample volumes.
8.4 Possible interference caused by substances contained in the sample can be recognized by using an internal standard as a control: in addition In a double determination using one sample solution, a difference of 0.005 to to the sample, blank and standard determinations, a further determination 0.010 absorbance units may occur. With a sample volume of v = 0.100 ml should be carried out with sample and assay control solution in the
and measurement at 340 nm, this corresponds to an ammonia concentration same assay. The recovery can then be calculated from the absorbance of approx. 0.4-1 mg/l. (If the sample is diluted during sample preparation, the result has to be multiplied by the dilution factor F. If the sample isweighed in for sample preparation, e.g. using 1 g sample/100 ml = 10 g/l, a 8.5 Possible losses during the determination can be recognized by carrying difference of 0.004-0.01 g/100 g can be expected.) out recovery tests: the sample should be prepared and analyzed withand without added standard material. The additive should be recovered The following data for the determination of ammonia have been published in quantitatively within the error range of the method.
CV = 0.88-1.16 % (ammonium chloride solutions) The reagents used in the determination of urea and ammonia are not (ammonium chloride solutions) hazardous materials in the sense of the Hazardous Substances Regulations, CV = 0.36-0.96 % (meat samples) the Chemicals Law or EC Regulation 67/548/EEC and subsequent alteration,supplementation and adaptation guidelines. However, the general safetymeasures that apply to all chemical substances should be adhered to.
After use, the reagents can be disposed of with laboratory waste, but local For separation of fat and for precipitation of the potassium perchlorate regulations must always be observed. Packaging material can be disposed refrigerate for 20 min. Afterwards filter. Discard the first few ml. Use the of in waste destined for recycling.
clear, possibly slightly turbid solution for the assay.
Calculate of the amount of urea and ammonia according to the aforemen- 10. General information on sample preparation
tioned calculation formula, whereby it must be multiplied with the volume In carrying out the assay: displacement factor K = 0.98.
Use clear, colorless and practically neutral liquid samples directly, or
after dilution according to the dilution table, and of a volume up to 2.000 ml;
12. Further applications
Filter turbid solutions;
The method may also be used in the examination of fertilizers, pharmaceuticals, Degas samples containing carbon dioxide (e.g. by filtration);
cosmetics, paper (Ref. 2.1) and in research when analyzing biological Adjust acid samples to pH 7-8 by adding sodium or potassium hydroxide
samples. For details of sampling, treatment and stability of the sample see Ref. 1.1-1.4.
Adjust acid and weakly colored samples to approx pH 7-8 by adding
sodium or potassium hydroxide solution and incubate for approx. 15 min;
Treat "strongly colored" samples that are used undiluted or with a higher
sample volume with polyvinylpolypyrrolidone (PVPP) - (e.g. 2.5-5 g/100 ml);
12.1 Determination of urea and ammonia in fertilizers
Crush or homogenize solid or semi-solid samples, extract with water or
Grind approx. 10 g of the sample and mix thoroughly. Accurately weigh dissolve in water and filter if necessary; approx. 100 mg of the homogeneous material into a 100 ml beaker and add Deproteinize samples containing protein with perchloric acid or with tri-
approx. 50 to 60 ml water. Adjust to pH 7-8 with diluted hydrochloric acid chloroacetic acid; (1 M) or in the case of acidic fertilizer with diluted sodium hydroxide (1 M).
Extract samples containing fat with hot water (extraction temperature
Warm on a heatable magnetic stirrer for approx. 10 min to 60-70°C. Allow to should be above the melting point of the fat involved). Cool to allow the fat cool, transfer quantitatively into a 100 ml volumetric flask and fill up to the to separate, make up to the mark, place the volumetric flask in an ice bath mark with water. Mix the solution and filter, if necessary. for 15 min and filter; Use 0.100 ml of the clear solution diluted, if necessary, for the assay.
Break up emulsions with trichloroacetic acid.
12.2 Determination of nitrogen after Kjeldahl-digestion
The determination of total nitrogen can be obtained via the ammonia The Carrez-clarification should not be used in the sample preparation
determination in a sample mineralized according to the Kjeldahl-method.
for urea / ammonia determination due to a too low recovery rate
Normally, the samples are to be incinerated wet (sulfuric acid). The ammonia, (adsorption of urea/ammonia).
formed from nitrogen, is determined according to the procedure as follows.
11. Application examples
Accurately weigh approx. 2 g of the ground and homogenized sample into a100 ml Kjeldahl-flask, add 20 ml sulfuric acid (specific gravity = 1.84 g/ml) Determination of ammonia in fruit juices
and approx. 30 mg catalyst mixture (e.g., acc. to Wieninger) or one Kjeldahl Add 0.5-1.0 g wet polyvinylpolypyrrolidone (PVPP) to 10 ml fruit juice (clear, tablet, heat for approx. 2-3 h until the sample is disintegrated (yellowish or turbid or colored juices) - when the sample volume is increased, neutralize, blue-greenish solution). Allow the sample to cool and carefully (protective if necessary, and fill up to 20 ml with water - in a beaker and stir for 1 min glasses) transfer quantitatively into a beaker filled with 600 ml ice-cold (magnetic stirrer). Filter sample solution immediately and use it for the assay.
water, while stirring all the time (magnetic stirrer, icebath). Neutralize with In the assay, only "blank ammonia" and "sample ammonia" are to be mea- approx. 60 ml KOH (10 M) (pH 6-8). Transfer the neutralized solution quanti- tatively into a 1 l volumetric flask, fill up to the mark with water and mix. If Determination of urea and ammonia in water (swimming-pool water)
necessary, filter the mixture (sometimes necessary after disintegration with Dilute sample solution according to the dilution table or use up to v = 2.000 ml Kjeldahl tablets); discard the first few ml. Use the solution diluted, if sample volume for the assay.
necessary, for the assay.
Determination of urea in milk
Mix 1 ml milk with 4 ml trichloroacetic acid (0.3 M). After approx. 5 min Nitrogen content of the sample (in %) centrifuge for separation of the precipitate (for 3 min, ca. 4000 rpm). Use 0.100 ml of the supernatant clear solution for the assay.
× d × v × 1000 × amount weighed [g] Determination of ammonia in milk
Mix 1 ml milk with 4 ml trichloroacetic acid (0.3 M). After approx. 5 min
A × 3.04 × 14.01 × 100 centrifuge for separation of the precipitate. Decant the supernatant and = × 1.00 × 0.100 × 1000 × amount weighed [g] neutralize with KOH (10 M) (dilution factor can be neglected due to the highconcentration of KOH), filter and use 1.000-2.000 ml sample solution for theassay.
12.3 Determination of urea and ammonia in fermentation samples
In the assay, only "blank ammonia" and "sample ammonia" are to be mea- and cell culture media
Place the sample (after centrifugation, if necessary) in a waterbath at 80°C Determination of ammonia in bakery products
for 15 min to stop enzymatic reactions. Centrifuge and use the supernatant Accurately weigh approx. 10 g of the minced sample into a homogenizer (diluted according to the dilution table, if necessary) for the assay.
beaker, add approx. 20 ml perchloric acid (1 M) and homogenize for approx.
Alternatively, deproteinization can be carried out with perchloric acid. See 2 min. Proceed as stated under "meat and meat products". Use at most the above-mentioned examples.
1.000 ml for the assay. Homogenize gelatinous agar media with water and treat further as In the assay, only "blank ammonia" and "sample ammonia" are to be mea- Determination of urea and ammonia in meat and meat products
Accurately weigh approx. 5 g of the homogenized sample (from a sample of
100 g, that has been ground and homogeneously mixed in a mixer) into a
homogenizer beaker, add approx. 20 ml perchloric acid (1 M) and
homogenize for approx. 2 min. Transfer the contents quantitatively with
approx. 40 ml water into a beaker. Adjust to pH 7.0 ( 7.5) first with
potassium hydroxide (5 M) and then exactly with potassium hydroxide (2 M).
Transfer the contents quantitatively with water into a 100 ml volumetric flask,
fill up to the mark with water, whereby it must be taken care that the fatty
layer is above the mark and the aqueous layer is at the mark.
Gutmann, I. & Bergmeyer, H. U. (1974) in Methoden der enzymatischen Analyse Höpner, Th. (1977) Enzymatische Methoden in der Wasseranalytik - Möglichkeiten und (Bergmeyer, H. U. Hrsg.) 3. Aufl., Bd. 2, S. 1842-1845, Verlag Chemie, Weinheim and Grenzen, Vom Wasser 49, 173-182
(1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U. ed.) 2nd ed., vol. 4, pp. 1794- Gerhardt, U. & Quang, T. D. (1979) Methoden zur Ammoniakbestimmung in Fleisch und 1798, Verlag Chemie, Weinheim/Academic Press, Inc., New York and London Fleischerzeugnissen, Fleischwirtschaft 59, 946-948
da Fonseca-Wollheim, F., Bergmeyer, H. U. & Gutmann, I., (1974) in Methoden der Erbersdobler, H. & Zucker, H. (1980) Harnstoff-Gehalt der Milch - ein Indikator der enzymatischen Analyse (Bergmeyer, H. U. Hrsg.) 3. Aufl., Bd. 2, S. 1850-1853, Verlag Proteinversorgung von Milchkühen, Kraftfutter 63, 10-12
Chemie, Weinheim and (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U. ed.) Wolfschoon-Pombo, A., Klostermeyer, H., Buchberger, J. & Graml, R. (1981) Harnstoff in 2nd ed., vol. 4, pp. 1802-1806, Verlag Chemie, Weinheim/Academic Press, Inc., New der NPN-Fraktion der Kuhmilch - Bestimmung, Vorkommen und Beeinflussung, Milch- wissenschaft 36, 462-466
Kerscher, L. & Ziegenhorn, J. (1985) in Methods of Enzymatic Analysis (Bergmeyer, H.
Barchietto, G., Cantoni, C., Frigerio, R. & Provera, D. (1984) Esame comparativo U., ed.) 3rd. ed., vol. VIII, pp. 444-453, Verlag Chemie Weinheim, Deerfield Beach/ deiprodotti di autolisi nella carne di maiale (Azoto non proteico, Urea, Ammoniaca), Conservazione degli Alimenti 3, 12-17
Bergmeyer, H. U. & Beutler, H.-O. (1985) in Methods of Enzymatic Analysis (Bergmeyer, Cheuk, W.L. & Finne, G. (1984) Enzymatic Determination of Urea and Ammonia in H. U., ed.) 3rd ed., vol. VIII, pp. 454-461, Verlag Chemie Weinheim, Deerfield Beach/ Refrigerated Seafood Products, J. Agric. Food Chem. 32, 14-18.
Kohler, P. (1985) Ringversuch für die enzymatische Bestimmung von Harnstoff in Untersuchung von Papieren, Kartons und Pappen für die Lebensmittelverpackungen Badewasser, Mitt. Gebiete Lebensm. Hyg. 76, 470-477
(gem. Empfehlungen XXXVI der Kunststoffkommission des Bundesgesundheitsamtes) Pasquier, J.-M. & Grandjean, L. (1985) Méthode de dosage de l'urée dans l'eau de Kapitel 8 (Methoden) Pkt. 3.4.2 (Ammoniak) und Pkt. 3.5.2. Harnstoff); März 1979 piscine, Trav. chim. aliment. hyg. 76, 464-469
Gombocz, E., Hellwig, E., Vojir, F. & Petuely, F. (1981) Deutsche Lebensmittel-Rundschau Buchberger, J., Weiß, G. & Graml, R. (1988) Untersuchungen zum Orotsäure- und 77, 9 (Harnstoff)
Harnstoffgehalt der Milch; I. Teil: Orotsäuregehalt, dmz deutsche molkerei-zeitung, Brautechnische Analysenmethoden, Band III, S. 597-599 (1982), Bestimmung von 37 1128-1133; II. Teil: Harnstoffgehalt, dmz deutsche molkerei-zeitung 38, 1167-1169
Ammoniak, Methodensammlung der Mitteleuropäischen Brautechnischen Analysen- Bartels, U. (1991) Die enzymatische Bestimmung von Ammonium im Niederschlags- kommission (MEBAK), herausgegeben von F. Drawert im Selbstverlag der MEBAK, Freising wasser, CLB Chemie in Labor und Biotechnik 42, 377-382
Nederlandse Norm NEN 6494 (Juni 1984) Water: Enzymatische bepaling van het gehalteaan ureum in zwemwater, (Water-Enzymatic determination of urea in swimming water) Urea assay control solution
The assay control solution serves as a control for the enzymatic determina- 3. Internal standard: tion of urea in foodstuffs and other materials.
The assay control solution can be used as an internal standard in order tocheck the determination for correct performance (gross errors) and to see whether the sample solution is free from interfering substances: Preparation of the assay control solution
Accurately weigh approx. 140 mg urea to the nearest 0.1 mg into a 1000 ml reaction mixture 2 volumetric flask, fill up to the mark with redist. water, and mix thoroughly.
Prepare assay control solution freshly before use. The assay control solution assay control sln.
may be frozen in portions.
Mix, and read absorbances of the solutions (A1) after approx. 5 min.
1. Addition of the assay control solution to the assay mixture: Continue as described in the pipetting scheme under "Procedure" . Follow Instead of sample solution the assay control solution is used for the assay.
the instructions given under "Instructions for performance of assay" and (The measurement fo the assay control solution is not necessary for calcu- the footnotes.
lating the results.) The recovery of the standard is calculated according to the following 2. Restart of the reaction, quantitatively: After completion of the reaction with sample solution and measuring of A2, add 0.050 ml assay control solution to the assay mixture. Read absorbance 2 × Asample + standard - Asample 3 after the end of the reaction (approx. 20 min). Calculate the concentration from the difference of (A 2-A3) according to the general equation for calcu- lating the concentration. The altered total volume must be taken intoaccount. Because of the dilution of the assay mixture by addition of theassay control solution, the result differs insignificantly from the result gotaccording to pt. 1.
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