Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016 Page 14361JMSCR Vol||04||Issue||12||Page 14361-14365||December 2016Utility of Osazone Test to Identify SugarsAuthorsDr Tejas Shah, MD, Dr Nikunj Modi, MD(Biochemistry), Dept. of Biochemistry, Shri M P Shah Govt Medical College, Jamnagar, Gujarat (India)Corresponding AuthorDr Tejas ShahMD (Biochemistry), Dept. of Biochemistry,Shri M P Shah Govt. Medical College, Jamnagar, Gujarat (India)Email: [email protected], Mob: 9879061781ABSTRACTCarbohydrates (sugars) are the most abundant organic molecules in nature. Sugars having reducing propertyreact with phenylhydrazine hydrochloride to form characteristic osazones (crystals). These osazones can becorrelated with clinical conditions like arabinose in autism, xylose in small bowel disease and galactose ingalactosemia. The study aimed to evaluate utility of osazone test to identify sugars by demonstratingcharacteristic osazone. Study was conducted at Biochemistry department, Shri M P Shah Govt. MedicalCollege, Jamnagar, Gujarat. We selected sugars like glucose, fructose, mannose, galactose, lactose, maltose,arabinose and xylose to demonstrate their osazone. All sugars were made available commercially. 2 grams%stock solution prepared of each sugars. Osazone test is performed for each sugar in the boiling water bath andnoted down the time for appearing of crystals. Then the shape of osazone of each sugar was examined undermicroscope. We observed that crystals were appeared at specific interval of time during boiling and coolingslowly after boiling. We found characteristic shape of osazone of sugars under microscope. Glucose, fructose,galactose and mannose formed needle shape osazone. Maltose formed sun flower shape osazone. Lactoseformed cotton-ball shape osazone. Arabinose formed dense ball needle shape osazone. Xylose formed fine butlong needle shape osazone.The study demonstrated characteristic shape of osazone of sugars by performingosazone test. This simple, cheap and less time consuming test can be used to identify and differentiate sugarsencountered in clinical practice.Keywords: Carbohydrates, phenylhydrazine, osazone.INTRODUCTIONCarbohydrates (Sugars) are defined chemically asaldehyde or ketone derivatives of the higherpolyhydric alcohols, or compounds which yieldthese derivatives on hydrolysis. They have awide range of functions, including providing asignificant fraction of the energy in the diet ofmost organisms, acting as a storage form ofenergy in the body, and serving as cell membranecomponents that mediate some forms ofintercellular communication. Sugars areclassified as simple and complex. Simple sugarsare called monosaccharides which cannot behydrolyzed into smaller compound. Complexsugars are disaccharides/oligosaccharides/polysaccharides which can be hydrolyzed tomonosaccharides. Sugars having free carbonylgroup (either aldehyde or ketone) are calledreducing sugars. The osazone reaction wasdeveloped and used by Emil Fischer to identifywww.jmscr.igmpublication.orgImpact Factor 5.244Index Copernicus Value: 83.27ISSN (e)-2347-176x ISSN (p) 2455-0450DOI: https://dx.doi.org/10.18535/jmscr/v4i12.14Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016 Page 14362JMSCR Vol||04||Issue||12||Page 14361-14365||December 2016aldose sugars differing in configuration only at thealpha carbon. All reducing sugars will formosazones with excess of phenylhydrazine whenkept at boiling temperature. Osazones areinsoluble. Each sugar will have characteristiccrystal form of osazones which can be used toidentify and differentiate sugars in biological fluidlike urine. By detecting such sugars, correlationcan be made with clinical conditions like (a)glucosuria in diabetes mellitus, fructosuria infructose intolerance/hereditary fructosuria, lactosein lactose intolerance, galactose in galactosemia(b) arabinose in autism/candidiasis and (c)xylose in small bowel disease/malabsorptionsyndrome.[7, 8] Recent study was done to evaluatecharacteristic osazones of uncommonlyencountered sugars. The present study wasaimed to evaluate utility of osazone test to identifysugars by demonstrating characteristic osazone.MATERIAL AND METHODSThe present observational study was carried out atBiochemistry department during July 2016 toAugust 2016. As this study did not involve anykind of intervention to living animals, InstitutionalEthics Committee permission was not taken. Weused commercially available monosaccharides anddisaccharides of analytical grade. We selectedcommonly encountered sugars like glucose,fructose, mannose, galactose, maltose, lactosealong with uncommonly encountered sugars likearabinose and xylose for study.Principle of osazone test:[9, 10]Reducing sugar reacts with one molecule ofphenylhydrazine hydrochloride (C6H5-NH-NH2)to form phenylhydrazone hydrochloride. Thiscomplex reacts once again with another moleculeof phenylhydrazine hydrochloride to give anintermediate keto derivative. This again reactswith one more molecule of phenylhydrazinehydrochloride to give corresponding osazone.This reaction is complete in 3 steps and consumes3 moles of phenylhydrazine. During reaction withmonosaccharides, additional phenyl hydrazine isconsumed in oxidizing the adjacent OH-group tocarbonyl group which then forms a second phenylhydrazone. Such bisphenyl hydrazones are calledosazones.Sugar + phenylhydrazine hydrochloride (sugarphenylhydrazone) + H2Osugar-phenylhydrazone + 2 (phenylhydrazinehydrochloride) osazone+C6H5NH2+NH3+ H2OProcedure:[11, 12]1. Preparation of stock solution; Weweighed 2 grams (gm) of each sugar andtook it into sterile glass beaker of 150 mlcapacity. Then added distilled water tomake total volume 100 ml. Thus each sugar stock solution (2 gm%) was prepared.2. Preparation of phenylhydrazinemixture: 1 part of phenylhydrazinehydrochloride mixed with 2 parts ofsodium acetate in a mortar.3. Osazone test: We took 5 ml of sugar stocksolution in sterile test tube. Then we added1 gm of phenylhydrazine hydrochloridemixture and 2 drops glacial acetic acid (tomaintain pH of the solution) into it. Thenafter mixing well, we put test tube intoboiling water bath and noted down thetime for appearance of osazone crystal.4. Microscopic examination: 2 drops ofsugar crystals were taken by sterile glasspipette and carefully placed on the glassslide. Then cover slip was placed above itand examined under high resolutionmicroscope. After clear vision of crystals,snapshots were taken for record.RESULTSThe present study was performed on 6monosaccharides (glucose, fructose, mannose,galactose, arabinose, xylose) and 2 disaccharides(maltose, lactose). When observation made,crystals were formed after specific time intervaleither by boiling or by cooling slowly afterboiling. When examining under microscope,characteristic crystals were found. They wereshown from figure 1 to 8. Table 1 showedcharacteristics of osazone studied.Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016 Page 14363JMSCR Vol||04||Issue||12||Page 14361-14365||December 2016Table 1. Characteristics of osazone studied Name of sugarShape of crystalTime required for crystalformation (min)Physical conditionsBoilingCooling slowly after boilingGlucoseNeedle6+++FructoseNeedle4+++MannoseNeedle10+++GalactoseBalls with thorny edge10+++ArabinoseDense ball needle15+++XyloseFine-long needle15+++MaltoseSun flower30–+LactoseCotton-ball30–+ + = appearance of crystal, ++ = intense appearance of crystal, – = no appearance of crystal.Figure 1. Needle shaped crystals (Glucose)Figure 2. Needle shaped crystals (Fructose)Figure 3. Needle shaped crystals (Mannose)Figure 4. Balls with thorny edge shaped crystals(Galactose) Figure 5.(Arabinose)Dense ball needle shaped crystals Figure 6. Fine-long needle shaped crystals(Xylose)Figure 7. Sun flower shaped crystals (Maltose)Figure 8. Cotton ball shaped crystals (Lactose)Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016 Page 14364JMSCR Vol||04||Issue||12||Page 14361-14365||December 2016DISCUSSIONCarbohydrates (sugars) are the most abundantbiological molecules. Glucose is the majormetabolic fuel of mammals (except ruminants)and a universal fuel of the fetus. It is the precursorfor synthesis of all the other carbohydrates in thebody. Biologically, the most important epimers ofglucose are mannose (epimerized at carbon 2) andgalactose (epimerized at carbon 4). Fructose hasthe same molecular formula as glucose but differsin that there is a potential keto group in position 2.Chemically, sugars are reducing compounds ifthey have free carbonyl group, and are sometimesknown as reducing sugars. This provides the basisfor a simple chemical test for glucose in urine inpoorly controlled diabetes mellitus.[14, 15] One ofthese tests is osazone test. Osazone test is thesimplest test to identify and differentiate reducingsugars in biological fluid like urine. It is routinelyperformed for glucose, fructose, mannose, maltoseand lactose. Monosaccharides are powerfulreducing agent in hot solution.[4, 9] We foundyellow, needle shaped crystals of glucose, fructoseand mannose while balls with thorny edge shapedcrystals of galactose (refer figure 1 to 4). Glucose,mannose and fructose due to similarities ofstructures form the same osazones. But since thestructure of galactose differs on C-4, that part ofthe molecule unaffected in osazone formation, itwould form a different osazone.[1, 16] Because offorming same shaped osazones, it is difficult todifferentiate glucose, fructose, and mannose. Buttime of appearance of crystals may have someutility in diagnosing diabetes and fructoseintolerance. Lactose and maltose are reducingdisaccharides because beta (14) and alpha(14) glycosidic linkages seen respectively sparecarbonyl carbon so that both form osazones.Lactose formed cotton-ball shaped crystals andmaltose formed sun flower shaped crystals onlyafter cooling slowly in present study (refer figure5 and 6). Osazones of reducing disaccharides aremore soluble in hot solution and don’t appearduring boiling. Hence they formed crystals slowlyon cooling the solution.[1, 16] We have alsoexamined uncommonly encountered monosaccharides like arabinose and xylose. Both are fivecarbon sugars found in plant gums, proteoglycansand glycosaminoglycans. They are constituent ofglycoproteins. We observed dense ball needleshaped and fine-long needle shaped crystals ofArabinose and xylose respectively (refer figure 7and 8). It is suspected that the arabitol producedby the yeasts in the gastrointestinal tract isabsorbed in the portal circulation, and is thenconverted into arabinose by the liver. It is notmetabolised endogenously and is eliminated bythe urine. Arabinose is often found to be raised inthe presence of intestinal candidiasis, and iscommonly found in autistic children. It issuspected that autistic children may havedeficiencies of one or more enzymes that areinvolved in the metabolism of pentoses. Highlevels of arabinose have been found linked toproteins in serum glycoproteins of serum ofschizophrenic patients and in children withbehavioural disorders. Xylose is absorbedunchanged by the duodenum and jejunum. Itsincomplete absorption allows for its possible useas a malabsorption test or to identify small bowelmucosal disease.[7, 8, 17] The findings of this studywere supported by Vinod BS et al study. Formore evaluation high pressure liquid chromatography (HPLC) is used nowadays which not onlydifferentiate but quantify such sugars. In theabsence of HPLC setup osazone testing can beutilized.CONCLUSIONWe demonstrated characteristic osazone ofcommonly and uncommonly encounteredreducing sugars. Osazone test have great utility toidentify and differentiate such sugars. Moreover,it is a simple, less time consuming and easilyperformed in laboratory by technician ascompared to HPLC.LIMITATIONSWe did not performed osazone test on urinesamples of patients. So it is difficult to evaluatethe factors which can affect the crystal formationin actual diseased condition.Dr Tejas Shah, MD et al JMSCR Volume 4 Issue 12 December 2016 Page 14365JMSCR Vol||04||Issue||12||Page 14361-14365||December 2016ACKNOWLEDGEMENTSWe are thankful to Mr. M H Rajapara and Mr.Manish Pandya, laboratory technicians,Biochemistry department, Shri M P Shah Govt.Medical College, Bhavnagar for their help andsupport during this study.Source of support: NoneREFERENCES1. Chatterjee MN, Shinde R, editors.Textbook of medical biochemistry. 8th ed.Ajanta offset & PackagingsLtd:Jaypee;2012:p.23-8.2. Ferrier DR, editor. Lippincott’s illustratedreviews Biochemistry. 6th ed. LippincottWilliam & Wlikins: Wolters Kluwer;2014:p.83-5.3. Internet source:http://www2.chemistry.msu.edu4. Vasudevan DM, Shreekumari S,Vaidyanathan K, editors. Textbook ofbiochemistry for medical students. 7th ed.Ajanta offset & Packagings Ltd, Jaypee;2013:p.69-74.5. Dennis LK, Eugene B, Anthony SF,Stephen LH, Dan LL, Larry J, editors.Harrison’s principles of internal medicine.16thed. McGraw-Hill; 2005:p.2319-23.6. http://www.labco.es/media/pdf/en_download_13.pdf. Arabinose test for theDiagnosis of intestinal Candidiasis: Labcoquality diagnostics.7. Christie DL. Use of the one-hour bloodxylose test as an indicator of small bowelmucosal disease. The Journal of Pediatrics.1978; 92(5):p.725–7288. Buts JP. One-hour blood xylose test: Areliable index of small bowel function. TheJournal of Pediatrics.1978; 92(5):p.729-33.9. Vinod BS, Santhi S, Krithika. Osazones ofthe Uncommonly Encountered ReducingSugars. International Journal of Interdisciplinary and Multidisciplinary Studies.2015 ;(2)9:p.24-29.10. Rafi MD. Textbook of biochemistry formedical students. 2nded. Universities press(India) Pvt. Ltd; 2014:p.30.11. Chary TM, Sharma HO, editors. Practicalbiochemistry for medical and dentalstudents. 1sted. Noida: Jaypee;2004:p.6-7.12. Chawla R. Practical clinical biochemistry:Methods and interpretations. 3rded.Noida:Jaypee; 2006:p.35.13. Voet D, Voet JG, Pratt CW, editors.Fundamentals of biochemistry: Life at themolecular level. 3rded. Wiley;2008:p.219.14. Rodwell VW, Bender DA, Botham KM,Kennelly PJ, Weil PA, editors. Harper’sillustrated biochemistry. 30th ed. Lange:McGraw Hill; 2015:p.152-55.15. Ngugi MP, Njagi JM, Kibiti CM,Ngeranwa JJN, Njagi ENM. Diagnosis ofDiabetes Mellitus. 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