International Journal of Scientific & Engineering Research, Volume 6, Issue 2, February-2015 148

ISSN 2229-5518

Absorption studies of Creatinine using

Kinetic Reaction method by optical

Interference wavelength filter

Sreedhar MALLETI

Research scholar,Dept of Instrument Technology, college of engineering, Andhra university

Visakhapatnam,Andhrapradesh,India

email: sonofsreedhar@gmail.com,09440976843

Saradadevi MALLETI

Assistant professor,Department of pharmaceuitical chemistry,sreenivasarao college of pharmacy,andhrauniversity,Visakhapatnam

email: sarada_pharma30@yahoo.co.in,9849948784

Dr.Varaprasad PLH

Professor, Dept of Instrument Technology ,college of engineering, Andhrauniversity

Visakhapatnam,Andhrapradesh,India

e-mail:plhpuvvada@yahoo.co.in,09959036321, Dr.Bhujangarao A

Professor and Head of thedept of Instrument Technology ,college of engineering, Andhrauniversity

Visakhapatnam,Andhrapradesh,India

e-mail:dr_abrao@gmail.com,09603741102

Abstract: Accurate measurement of Creatinine is very essential in order to pre diagnosis the renal functioning of human body. In this paper we introduced the measurement of creatinine by means of studing its equivalent absorption with the interaction of wavelength of light using kinetic reaction method with the help of photometer.

Keywords: absorption creatinine kinetic reaction photometer

1.1 INTRODUCTION

Creatinine reacts with Picric Acid in an alkaline medium to form an Orange coloured complex [1-
5]. The rate of formation of this complex is measured by reading the change in absorbance at
505 nm in a selected interval of time and is proportional to the concentration of Creatinine. The reaction time and the concentration of Picric Acid and Sodium Hydroxide have been optimized to avoid interference from keto acids.
Creatinine + Picric Acid Orange coloured complex
Breakdown product of ‘creatinine phosphate’ in muscle produces at constant rate by the body and filtered out of blood by kidneys poor filteration of urine results rise in creatininelevel Calculation of creatinine clearance is required in the urine and blood.

1.2.REAGENTS COMPOSITION (TABLE 1)

1.3.REAGENT STORAGE AND STABILITY Prior to reconstitution

Unopened Reagents 1,2 are stable at Room
Temperature (15 – 300c) and reagent 3 is stable at
2-8° C until the expiry date mentioned on the container label.

After reconstitution

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The “Working Reagent” is stable for 7 days at 2-80
c.

Experiment Technical specifications and instrumentation

Sample volume: 70µl serum;1 1 precision µl
Reaction temperature: 37 ̊ C
Cuvettes size: 5X6X25; 5mm opticl length; Reaction volume: 180-500 µl
Photometric system: interference filters of with static –fibre optics
Lamp: tungsten-halogen lamp Environment requirements: 15 ̊ C-30 ̊ C Humidity: 35%-80%
Atm.pressure: 800hPa-1060 hPa

a)Light source : Tungsten halogen lamp

b)Interference filter[6-7]:it relies on optical interference to provide narrow band of radiation. some times these are also called as fabry perot filters. The interference filters are available for the ultraviolet, visible and well into the infrared region. An interference filter consists of a transparent dielectric(frequently calcium fluoride or magnesium fluoride)that occupies between two semi transparent materials. The thickness of the dielectrical layer is carefully controlled and determines wavelength of the transmitted radiation. when a perpendicular beam of collimated radiation strikes this array, a fraction passes through the first metallic layer and the remainder is reflected. The portion that is passed undergoes a similar partion when it strikes the second metallic film. if the reflected portion of this second interaction is of the proper wavelength, it is partially reflected from the inner side of the first layer in phase with the incoming light of the same wavelength. The result is that this particular wavelength is rein forced, and most other wavelengths, being out of phase, undergo destructive interference.

d)lens system and photometer: This system consists of 9 optical paths with interference filters

Wavelengths:340 nm,405nm,450nm,510nm,546nm,578nm,630nm,67
0nm,700nm Half band width:≤12nm
Measurement range:0.1-4.0 Abs
Lamp: 12 V 50 VA tungsten-halogen

1.4 METHODOLOGY

It is purely based on kinetic reaction. The reaction velocity is not related to the substrate concentration and remains constant in the reaction process. As a result, for a given wavelength ,the absorbance of the reacting liquid changes evenly, and the change rate(∆A/min)is proportional to the activity or concentration of the subject, which is usually the enzyme.

Fig.1.1.kinetic reaction method
In fact, it is impossible for the substrate concentration to be large enough, and the reaction will no longer a zeroth order reaction when the substrate is consumed to ascertain degree.therefore,the theory only stands within certain period. In addition the reaction can become steady only after a certain period. In addition, the reaction can become steady only after certain period because the reaction is complicated at the beginning and there are miscellaneous reactions due to the complex serum compositions. All reagent.
As shown in the above figure1.1,t1 is the time when the reagent is added.t2 is the time when the sample is added. From t3 the reaction becomes steady.tn is the time to stop testing the reaction.t3- t2 is the time delay, and tn-t3 is the monitoring time.

c) Absorbance and Response

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When a parallel monochromatic light beam whose intensity is Io goes through a flow cell(whose length is L) containing a solution (whose concentration is C),some photons are absorbed, and the intensity is attenuated from Io to I t,so the absorbance A of this solution is:
A=-Log It/I0
Where, It/I0= transmittivity
For the analyzer, the response(R)is defined as the absorbance change before and after the reaction,or the absorbance change rate during the reaction process.

1.5 CALCULATING THE RESPONSE OF THE KINETIC REACTION

single wavelength (for single –reagent )

As shown in the above figure1.1,
t1- is the time when the regent(volume) is added
t2- is the time when the sample(volume:S)is added. t3 -is the time from which the reaction becomes
steady
tn- is the time when the test result on the reaction is stopped (t3-t2) -is the time delay
(tn-t3)- is the reaction n time
The response is equal to the slope of the linear section between t3 and tn.

1.6 EXPERIMENTAL RESULTS[5](TABLE 2)

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1.8RESULTS AND CONCLUSION (TABLE:04)

(Primary wave length filter)

(secondary wave length filter) (TABLE:05)

Calculations

Primary wave length filter: Tn-T3=48.203 sec Absorbance:An-A3=28

Response or the Slope of the linearization portion is=[(391-(-381)]/(362.609-278.515)
=-10/84.094=0.11 Abs per sec

secondary wave length filter:

1.7 GRAPH(ABSORPTION VS TI ME IN SECONDS)

30000

Tn-T3=24.073sec
Absorbance :An-A3=6
Slope of the linearization portion is=[(-621-(-
629)]/(567.171-507.156)=8/60.015=0.133 Abs per

A b 20000 b a 10000 s n

o c 0

r e -10000

time

P.Wave length

P.Absorba nce

sec
(TABLE:06)(comparision of two filters)

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Hence it is concluded that by using the kinetic eaction method the response of the secondary wavelength filter to the creatinine of the human serum is much better than primary wave length filter.

References

i.Murray R.L: Non Protein nitrogen compounds, in Clinical chemistry: Theory, Analysis and Co Relation, Kaplan L.A. and Pesce A.J; Eds.

C.V. Mosby, Toronto, 1984. P1230-1268.

ii.Bowers L.D; Clin. Chem; 26,p551-556(1980)Young D; In

Effect of Preanalytical variables on Clinical Laboratory Tests,

2nd ed; AACC Press, Washington, 1997,p4-494

iii.Newman D.J; price C.P; Renal Function and Nitrogen Metabolites, InTietz Textbook of Clinical Chemistry, 3rd ed., Burtis C.A. and Ashwood E.R., Eds. W. B. Saunders, Philadelphia,1999, p 1204-1264.

iv. Kaplan A., Lavernal L. S., The kidney and tests of Renal Function, In Clinical Chemistry: Interpretation and Techniques, 2nd ed., Lea and Febiger, Philadelphia, 1983, p 109-

142.

v. Mathew TH. Australasian Creatinine Consensus Working Group. Chronic kidney disease and automatic reporting of estimated glomerular filtration rate: a position statement. Med J Aust. 2005;183:138–41. [PubMed].

vi.. Levey AS, Coresh J, Greene T, et al. Expressing the MDRD study equation for estimating GFR with IDMS traceable (gold standard) serum creatinine values (abstract) J Am Soc Nephrol. 2005;16:69A.

vii.. NKDEP Website (accessed 11th October

2006).http://www.nkdep.nih.go/ viii.. Jaffe M. Uber den niederschlag, welchen pikrinsaure in normalen hrn erzeugt und uber eine neue reaction des kreatinins. Z Physiol Chem.

1886;10:391–400.

ix.. Haeckel R. Assay of creatinine in serum, with use of

Fuller’s Earth to remove interferents. Clin Chem. 1981;27:179–

83.[PubMed]

x.. Cook JG. Factors influencing the assay of creatinine. Ann

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xii. sample data and reaction data (sample type: human serum) andhra medical college,VisakhapatnamA,P state,india.

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Author’s Profiles

Sreedhar.Malleti is presently a research scholar of Department of Instrument technology,Andhrauniversity,visakhapatnam. He worked as executive(instrumentation) in food industry for 2years.he was also the former associate professor and head of the department in Sir C.R.Reddycolllegeofengineering,eluru,westGodavari(dt),andhrapra desh,India.

Ms.Saradadevi malleti is presently working as Assistant professor in sreenivasarao college of pharmaceutical sciences affiliated Andhra university,Visakhapatnam,Andhra Pradesh,india.She was submitted her thesis report on chalcones synthesisin the year 2013 and awaiting for the award of the same. she did her post graduation in pharemaceutical sciences in Andhra university, Visakhapatnam,

Dr.A.Bhujangarao is currently a Professor and Head of the Department of Instrument technology,Andhrauniversity,Visakhapatnam,Andhrapradesh,India

.rauniversity,Visakhapatnam,Andhrapradesh,India.He Obtained his PhD in Instrument technology from Andhra university. He had published several national and international journals. He has guided some of research scholars. He was also worked as Chairman for Board of studies for Instrumentationengineering,Andhrauniversity,visakhapatnam.

Dr.P.L.H.Varaprasad is currently retired Professor and Head of the Department of Instrument technology of Andhra university, Visakhapatnam, Andhrapradesh, India. He Obtained PhD in Instrument technology of Andhra University. He had published several national and international journals. He has guided some of researchscholars.he is very much interested in the area of thin film technology. He was also worked as Chairman for Board of studies

for Instrumentation engineering of Andhra University.

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