ASIAN J. EXP. BIOL. SCI.

VOL 2(1) 2011: 28-33

Society of Applied Sciences

ORIGINAL ARTICLE

Quantitative and Micro Determination of Urine Sugar in Experimental Rats: Modified Anthrone Method*
R. Kannadhasan*
Asst.Professor, Dept. of Pharmacology and Toxicology, School Of Pharmaceutical Sciences, Vels University, Chennai-600 097.Tamil nadu, India.
ABSTRACT The periodical quantitative determination of glucose in urine experimentally in lower animals (viz., rats and mice, which excretes only a few mililitres) were found to be tedious using Benedict's tritrimetric method, or other methods like Shaffer-Hartmann iodimetric method, where more quantity of urine is used. At this condition all papers those reported previously in lower animals for urine sugar analysis only showed th after 24 hour readings. In this case the present study favours the urine sugar analysis for a regular interval period of 0-24 hrs using anthrone reagent. Anthrone reacts with few mililitres of diluted urine sample forming furfural ring complex, indicated by the change of colour, whose absorbance read at 620 nm. In this study we performed urine sugar analysis of diabetic rats using anthrone reagent, which is then compared with Standard glucose solutions. The results showed that the urinary glucose estimation with anthrone reagent was found comparable near with the values of Benedict's reagent (p=ns). Our results concludes that anthrone method of urinary glucose estimation is quite easier and predictable in normal and diabetic induced lower animals for a regular interval period of 0-24hr GTT study with reproducible point of accuracy. KEYWORDS: Modified anthrone method, Urinary glucose dilution (g/ml), STZ diabetic, Alloxan diabetic

INTRODUCTION The use of anthrone for the analysis of carbohydrates was first suggested by Dreywood[1], in 1946, who confirmed its specificity for carbohydrates. Morris [2] and Morse [3] adapted Dreywood's qualitative test to quantitative analysis of carbohydrates in samples. For quantitative determination of carbohydrate sample, 2gm of anthrone is dissolved in 1 litre of 95% sulfuric acid for anthrone reagent preparation. 4 or 5 ml of the sample to be analysed made to react with 8 or 10 ml of the reagent and mixed thoroughly by swirling. After 10mins the sample was subjected for analysis in a visual colorimeter at 540nm (green) or 620nm (red) filter. By this way they studied the specificity of anthrone for all the carbohydrates viz., pure mono-, di- and polysaccharides as well as with all samples of dextrin, dextrans, starches, plant polysaccharides, gums, glycosides and the acetates of mono-, di- and polysaccharides and also noted the significance of heat of the reaction. Seifter et al [4] studied the rate of liberation and dissipation of heat and suggested that prolonged heating may cause the depth of the color to fall off and to avoid such circumstances they traced with some identical glucose samples kept in test tubes were made to react with anthrone under submerged condition in cold water, followed by 10 mins heating and again cooling and read absorbance at 620nm. One of the major difficulties in the use of the anthrone method is the darkening of the anthrone-sulfuric acid reagent with time. To overcome this, it is necessary to prepare fresh reagent each day and make a new standardization curve or regression equation for each series of determinations. Chemistry of the anthrone reaction was already reported by several investigators. Anthrone, C6H4COC6H4CH2, is a derivative of anthraquinone. It is produced by the catalytic reduction of anthraquinone by hydrochloric acid in the 28 ASIAN J. EXP. BIOL. SCI. VOl 2 (1) 2011

18. Quantitative and Micro determination ...................................................................................... Modified anthrone method R. Kannadhasan

presence of metallic tin. It may exist in the keto or enol forms, known, respectively, as anthrone and anthranol. Hurd and Isenhour [5] and Wolfrom et al [6] postulated the possible mechanism of ring formation of carbohydrates and its derivatives with anthrone in the presence of the strong mineral acids. The breakdown of glucose to 5(hydroxymethyl)-2-furaldehyde represents dehydration with either double bond or ring formation which is indicated by green colour produced. This development of the green dye in the anthrone-carbohydrate reaction depends on 5(hydroxymethyl)-2-furaldehyde, or a similar furfural compound formed, by the action of the sulfuric acid on the carbohydrate used[7]. Momose et al [8] suggested from the chromatographic evidence of the benzene extract of the dye, the molecular weight of one of the main dyes to be approximately 530, with molecular formula, C47H30O3 (1,2,5,- or 1,3,5,trianthronylidenepentane). Therefore 3 moles of anthrone react with 1 mole of glucose in the following manner: 3C14H10O + C6H12O6 C47H30O3 + 5H2O + CH2O On consideration with above facts, few modifications were done in our study such as 1. Standardization of the reagent prepared in order to get concordant results. 2. Dilution of urine sample. 3. Making of anthrone reagents in aspect to react with diluted urine sample. 4. Stability of the reagent. MATERIALSAND METHODS Chemical and reagents Anthrone, Streptozotocin and Standard Glucose (Analytical Grade) were purchased from Sigma laboratories, Mumbai. Sulfuric acid (97%) purchased from Merck Co., andAlloxan monohydrate from SRL laboratories, Mumbai. Animals In bred colony of Male Sprague dawley rats (200 250g B.W) of Dr.C.L.Baid Metha college of Pharmacy were used. Preparation of anthrone reagent 2.5 gm of Anthrone was taken in a petri dish and 4ml of equal quantity of benzene and light petroleum ether were added and made to evaporate. The solution gets evaporated leaving the floc crystals of pure anthrone, collected and used for preparation of the reagent. 2 per cent of anthrone was prepared (recrystallized from benzene and light petroleum ether) in reagent grade ethyl acetate and kept stored in brown coloured bottles. This may be used as a stock for the routine analysis of urine sugar, which is stable for more than 3 months. Standardization ofAnthrone reagent Serial dilutions of 0, 20, 40, 60, 80 and 100 g/ml of Standard glucose solution were made. From each series 2ml of glucose solutions were transferred into the test tubes containing 5ml of conc. sulfuric acid, kept in ice water to minimize the turbulence produced in reaction with anthrone. 0.5 ml of anthrone-ethyl acetate reagent was carefully layered over the inner side of the test tubes and gentle swirling used to hydrolyze the ethyl acetate, giving floc of anthrone which redissolves and more rapid swirling mixes the contents of the test tubes. After proper mixing the contents were transferred to a 100oC boiling water bath for 10 minutes, followed by 4oC, for 5 minutes and to prevent o condensation of moisture on the optical tubes reading, 5 minutes in water bath at 20 C. Induction of Diabetes The animals for the study were housed in 12 hours day and night cycle for 3 days. Prior to the induction of diabetes animals were made fasted for a period of 16 hours. STZ (at a dose 45 mg/kg/i.p.,) and alloxan monohydrate (150 mg/kg/i.p.,) were administered for their respective groups. 1% glucose solution was given orally kept for 24 hours to avoid any hypoglycemic shock. On the next day the blood samples were analyzed using one touch glucometer. Blood glucose level 200mg/dl for STZ induction (Type 2) and 300 mg/dl for alloxan induced (Type 1) diabetic rats were chosen for the study. All experiments were carried out according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).The study was approved by the Institutional Animal Ethical Committee (Ref No: IAEC/41/CLBMCP/131; 2007-2008). Modified method of Urine sugar estimation Animals of both sexes were segregated in to 3 groups, one serves as a normal control and other two were diabetes induced (with Streptozotocin-45mg/kg/i.p., and Alloxan.H2O-150mg/kg/i.p., respectively). Before starting the experiment the animals were over night fasted (4 hours fasting in case of regular days check up) for 16 hours. Glucose loaded 2g/kg in 5ml of water respectively to all groups after initial BG reading following 1, 2, 4, 8 and 24 hours BG ASIAN J. EXP. BIOL. SCI. VOl 2 (1) 2011 29

18. Quantitative and Micro determination ...................................................................................... Modified anthrone method R. Kannadhasan

1ml of urine sample from each regular intervals were 2 fold diluted with distilled water and from this diluted urine, 1 ml was taken in a test tube containing 5 ml of 97 % Sulfuric acid kept in cooling water bath. 0.5 ml of anthrone was carefully layered over the sides of the test tube with gentle swirling so as to mix the contents without any bumping. This reaction mixture was then kept in 100o C boiling water bath for 10 mins, followed by 4o C for 5 mins. To avoid any moisture condensation in the cuvette the reaction mixture kept in 20o C for 5 mins before reading. The absorbance read for blank, test samples at 620 nm. Simple conversion formulae Concentration yield (g/ml) C =AS-AB x 100 WhereAS andAB are absorbance of the sample and blank used. Using dilution factors, DC = C x 100 (x 2 if 2ml of sample is used) DC diluted concentration. Conversion of g/ml to g/dl, g/dl = DC x 100 1000000 [Note: This modified procedure varies from the previous method by the substitution of direct urine sample instead of plant carbohydrate material and there was a simple conversion formula proposed by the author, for the conversion of urine sugar from g/ml to g/dl. Careful addition of Hcl was made under cool condition so as to avoid darkening of the sample before reading]. Stability of the reagent used To check the stability of anthrone reagent the following tests were made. A series of glucose solutions, each containing 0 (blank, distilled water), 20, 40, 60, 80, and 100 g/ml were reacted with the reagent and sulfuric acid (glucose solution, 1 ml; reagent, 0.5 ml; sulfuric acid, 5 ml.), relying solely on the heat of reaction of the acid and water to produce the green dye. These samples were allowed to stand 10 minutes following mixing and were then read in the colorimeter for the absorbance. The percentage transmittance of the contents was studied for regular interval of days. Decrease in % transmittance will implicate the losing stability of the reagent. Statistical analysis Statistical analyses were done using Graph pad prism software, Version 4. The standardization of anthrone reagent and its stability were analyzed using Two way anova followed by Bonferroni's post test comparing single column of values. Values are expressed as mean SEM of 3 replicates. 24th hour of urinary sugar of anthrone and benedict's quantitative test were analyzed with unpaired't'- test, values are expressed as mean SEM (n=6). RESULTSAND DISCUSSION Standardization ofAnthrone reagent From the Table 1, it was observed that the serial glucose concentration by anthrone method has no changes as compared with that of the benedict's quantitative method (p=ns). Moreover the accuracy was studied through the standardization of the anthrone reagent (Fig. 1). Modified method of Urine sugar estimation From the table 2, the urine sugar concentration in normal, STZ induced diabetic and Alloxan induced diabetic groups were found comparable near to the values of 24th hr reading of Benedict's quantitative method (p=ns) and it was also observed that the changes in urine sugar concentration is irrespective of the volume excreted. Stability of the reagent From the table 3, the percentage transmittance of spectrum light was found to be similar in the 90th day of observation as compared with that of the 1st day transmittance. And there was a remarkable change in the transmittance studied at 120th day of observation (Fig.2).

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ASIAN J. EXP. BIOL. SCI. VOl 2 (1) 2011

18. Quantitative and Micro determination ...................................................................................... Modified anthrone method R. Kannadhasan

Table 1 Standardisation of Anthrone reagent

Concentration (g/ml) Anthrone method: Required quantity of sample (g/ml) Absorbance (A) Concentration yield (g/ml) C = AS-AB x 100 0 (Blank) 20 40 60 80 100 0.0099 0.0002 Benedicts Method: Required quantity (ml) Conversion of C (g/dl) used from the Stock Solutions Quantitative (10ml) C = g/dl 0.2257 01588 0.4403 0.0065 0.6360 0.0334 0.8140 0.0205 1.120 0.0704 Qualitative (1ml)

g/dl = C x 100 x 100 100 000 0 0.1944 0.0053 0.3957 0.0100 ns 0.5961 0.0101 ns 0.7921 0.0069 ns 0.9924 0.0130 @

Green (<0.5g/dl) Green (<0.5g/dl) Green precipitate (>0.5g/dl) Yellow (>0.5 to 1g/dl) Yellow Precipitate (1 to 1.5g/dl)

0.2043 0.0052 20.43 0.5239 0.4057 0.0098 0.6060 0.0098 0.8020 0.0067 1.002 0.0129 40.57 0.9770 60.60 0.9815 80.20 0.6658 100.2 1.284

Values are Mean SEM of 3 replicates followed by Two way Anova using Bonferroni's Post test comparing single column of values. * = p<0.001; @ = p<0.01; # = p<0.05; ns = non significant.

Table 2 Modified Urinary Glucose estimation using Anthrone ethyl acetate reagent
Groups Methods BG (mg/dl) Anthrone Benedicts quantitative STZ diabetic induced BG (mg/dl) Anthrone Benedicts quantitative Alloxan diabetic induced BG (mg/dl) Anthrone Benedicts quantitative UG UG Parameters Blood Glucose (mg/dl) &Urinary glucose (mg/dl) concentrations at regular interval of time (Hours) 0 hr 1 hr 2 hr 4hr 24 hr 67.670 3.169 107.300 1.801 86.170 1.887 67.670 2.860 0.0012 0.0004 0.0017 0.00035 0.0010 0.0004 0.0020 0.0002 ns (2ml) (3.5) (6ml) (15ml) 0.0021 0.0001 111.8 2.182 a 139.200 3.790 296.5 8.782 0.0255 0.0001 (3ml) 330.700 15.090 0.2458 0.0135 (3.2ml) 266.5 17.68 0.6700 0.0291 (6ml) 308.0 6.962 1.245 0.0303 (5.2) 204.2 16.64 1.099 0.0273 (17.2ml) 263.5 11.76 1.671 0.0346 (11.6) 131.0 7.698 1.274 0.0410 (20.3ml) 1.262 0.0510 ns 138.0 6.748 1.975 0.0679 (24.2ml) 2.047 0.0750 ns

Normal control

UG

n= 6, Values are expressed as mean SEM. Comparison made at 24 hr of urinary glucose excretion using unpaired't'- test. * = p<0.001; @ = p<0.01; # = p<0.05; ns = non significant.

th

Table 3 Stability of the reagent for a regular interval of days

Concentration (g/ml) 0 20 40 60 80 100

Percentage Transmittance (% T) Day 1 Day 90 99.33 0.0003 96.73 1.146 ans 79.57 0.0053 76.94 0.190 ans 59.43 0.0098 57.83 1.516 ans 39.40 0.0098 38.49 0.616 ans 19.80 0.0067 18.60 0.417 ans -0.20 0.0128 1.67 0.567 ans

Day 120 65.90 0.306 b* 46.67 1.084 b* 26.47 3.246 b* 24.93 2.774 b* 10.60 4.588 b@ 4.97 3.110 bns

Values are expressed as mean SEM of 3 replicates followed by Two way Anova using Bonferroni's Post test comparing single column of values. a = Day 1 Vs Day 90 b = Day 90 Vs Day 120

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18. Quantitative and Micro determination ...................................................................................... Modified anthrone method R. Kannadhasan

F ig . 2 s h o w in g th e % T r a n s m itta n c e o f th e a n th r o n e r e a g e n t o n s ta b ility te s t

Fig. 2 showing the % Transmittance of the anthrone reagent on stability test

% T r a n s m it ta n c e

120

N o .o f D a y s

90

D ay 1 D ay 90 D ay 120

60

30

0 0 25 50 75 100

C o n c e n tr ati o n (m g /m l )

DISCUSSION The standardization of the reagent favours the point of accuracy of the reagent were it stands for its nature of forming anthrone-glucose complex or furfural ring [5,6]. The periodical changes in the urine sugar concentration in normal, STZ induced diabetic and Alloxan induced diabetic groups which is inversely proportion to the blood glucose concentration and volume of urine excreted. i.e., Increase in blood glucose concentration, there will be a decrease in urine sugar irrespective of the urine excreted. 32 ASIAN J. EXP. BIOL. SCI. VOl 2 (1) 2011

18. Quantitative and Micro determination ...................................................................................... Modified anthrone method R. Kannadhasan

The appearance of glucose in urine is reflected in the concept of a renal threshold for glucose excretion. The concept of a renal threshold for glucose excretion is propagated with the threshold specified at ~10 mmol/L [9]. According to this concept, no glucose should be detectable in urine at subthreshold blood glucose levels. Hence in this study glucose of 2g/kg in 5ml of water to each animal favours the urinary glucose excretion above the subthreshold level. There was no significant difference in the urine sugar level at 24th hr of both anthrone and benedicts quantitative method (comparison made at 24th hr as benedict's method requires around 10ml of urine for the assay). The 2 fold dilution of the urine prevents the possible hindrance of the impurities present in the urine during reaction with anthrone reagent [10]. The stability of the reagents stands for 3 months from the date of preparation where the point of accuracy will remain still. CONCLUSION From the above data's, it was very clear that, the modified method of urinary glucose estimation using anthrone reagent was quite easier and reproducible. This method of urinary glucose estimation is very much useful in assessing the GTT of any form of drugs in experimental diabetic rats. It was also proved that the periodical changes in urine sugar with that of the blood glucose concentration is irrespective of the quantity of urine excreted. The stability of the reagent prevents the laborious work of preparation and standardizations. In conclusion it can be stated that the anthrone method is very well suited for the accurate determination of glucose in the urine of diabetic animals. The rapidity and simplicity of the method allows large series of samples to be determined, even when the periodical 0-24 hr glucose excretion and the urinary glucose concentration are widely diverging. REFERENCES
[1]. [2]. [3]. [4]. Dreywood, R. (1946). Qualitative Test for Carbohydrate Material. In: Ind and Eng Chem. Anal ed., 18(8): p499. Morse, E,E., (1947).Anthrone in Estimating Low Concentrations of Sucrose. In: Ind. and Eng. Chem. Anal ed., 19:1012-1013. Morris, Daniel. L., (1948) Quantitative Determination of Carbohydrates with Dreywood'sAnthrone Reagent. Science, 107: 254 - 255. Seifter Sam, Dayton Seymour, Novic, B and Muntwyler Edward, (1950). The Estimation of Glycogen with the Anthrone Reagent. Arch Biochem, 25:191-200. [5]. Hurd, Charles. D and Iesnhour, Lloyd. L. (1932). Pentose Reactions. I. Furfural Formation. JAm Chem Soc., 54:317-330. [6]. Wolfrom, M.L, Scheutz, R.D and Cavalieri, Liebe. F., (1948). Chemical Interactions of Amino Compounds and Sugars. III. The Conversion of D-Glucose to 5-(Hydroxymethyl)-2-Furaldehyde. JAm Chem Soc., 70: 514-517. [7]. Sattler, Louis and Zerban, F.W., (1948). The dreywood anthrone reaction as affected by carbohydrate structure. Science, 108 (2800): 207. [8]. Momose Tsutomu, Ueda Yo, Sawada Kei and Sugi Ayako. (1957). OrganicAnalysis. VIII. Reaction Mechanism of Anthrone with Sugars. Pharm Bull, 5(1): 31. [9]. Klaus Rave, Leszek Nosek, John Posner, Tim Heise, Kerstin Roggen and Ewoud-Jan van Hoogdalem (2006). Renal glucose excretion as a function of blood glucose concentration in subjects with type 2 diabetesresults of a hyperglycaemic glucose clamp study. Nephrol Dial Transplant, 21: 2166-2171. [10]. Zweens, J and Bouman, P.R., (1968). Simple and Accurate Determination of Urinary Glucose Excretion with Anthrone Reagent. Diabetologia., 4: 278-280.

Corresponding Author: R. Kannadhasan, Asst.Professor, Dept. of Pharmacology and Toxicology,School Of Pharmaceutical Sciences, Vels University, Pallavaram, Chennai -600 117.Tamil nadu, India. Email: ramkhannasolutions@gmail.com

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