DISCUSSION AND CONCLUSIONS

Transcription

1 Chapter V DISCUSSION AND CONCLUSIONS In this chapter the findings of the present investigation on human renal excretory fluid i.e. urine is discussed. The parameters and spectroscopic analysis is discussed. Important conclusions are made with regards to the experimental results of the present investigation.

2 195 The urine is a bio fluid; the metabolic wastes in the human body are excreted in the form of urine. The urine has a significant place in medicine and pathology to social customs and agriculture. The onefifth of world population is suffering from urine related problems, such as kidney malfunction called nephrological problems and urological problems such as kidney stone formation. In mythology, it is considered as untouchable in some religions. Some argue that there is a strong possibility of there being several beneficial substances not yet known to science. The auto-urine therapy is an ancient therapy which believes that the intake of early morning urine supplements the essential nutrients and strengths the body. In agriculture the stored urine is used as a fertilizer. The urine is studied extensively by pathologist, physiologist, biochemists, and bio-medical engineers, but it has not drawn much attention of physicists. The application of concepts, principles and techniques of physics for the solution of problems in biology at different levels of complexity to get internal picture is drawing the attention of many researchers. The aim of the thesis is to develop these ideas at molecular level by studying the biophysical properties of human urine at different physiological conditions. Also, to examine the use of Physical properties of urine in medical discipline as the complimentary investigation to existing pathological and biochemical approaches. For this, the physical parameters, which are rheological (viscosity and surface tension), Optical (refractive index) and electrical (electrical conductivity and ph) of renal excretory fluid the urine have

3 196 been selected. The most interesting feature of the thesis is the application of IR spectroscopy to urine for the disease analysis. The study has been made considering the following aspects: 1. To determine physical parameters of urine of age group ranging from 22 to 64 years at the condition of collection morning first urine and random collection. 2. To evaluate the influence of Glucose, Urea, Albumin, Bilirubin and creatinine on physical properties of urine in order to investigate biophysically the diseases like diabetes, jaundice etc. Also, to assess which physical parameter is sensitive to a particular compound with which urine is treated. 3. Can spectroscopic analysis be complementary or best substitute to tedious and time consuming biochemical procedures? 4. To investigate pathological urine of the patients suffering from renal complications mostly due to Diabetes mellitus and also to evaluate glucose, urea, albumin and creatinine through sensitive physical parameters and IR analysis. As is known the urine is an important means of elimination of waste materials in the body. It is formed after filtration of blood in the glomerulus called Glomerular filtrate, reabsorption of glucose, amino acids and ions, and secretion of some drugs. The end product and the major constituents of urine are urea, creatinine and ions of sodium, chlorine, phosphate and potassium. The normal value of urine excreted by an adult is about 1250 ml per day. The abnormal decrease

4 197 or increase in urine volume indicates kidney malfunction. The specific gravity which indicates the solute concentration is the basic parameter in pathology which has a normal value and colour of urine is amber or lemon colour for healthy adult. The pathological test of urine includes physical, chemical and microscopic examination. In the present study specific gravity was found as the ratio of density of urine to that of pure water by using a specific gravity bottle up to accuracy of The mean value of urine specific gravity (USG) for first morning collected is ± and that of randomly or spot collected urine is ± Thus the average value of USG is It is observed that the first morning urine is concentrated of the day as the excretion of urine urea and electrolytes such as sodium, Na +, Cl -, potassium are eliminate only through urine because of the body was in rest. The urine samples are collected from a healthy donor in age group of 22-26, and There is no relation found between USG and age. Since concentration and composition of urine changes continuously during the course of 24 hour, according to water intake and activities before the test. It is important to regulate some aspects of urine collection, like time of collection, length of collection period (2hr, 12hr, 24hr and mid day clean catch), dietary intake and medicinal intake and method of collection to represent patient s truly metabolic state. The time of the day when specimen is collected influences the test result and findings. The first morning specimen is valuable, as it is relatively free of dietary

5 198 influence and change caused by physical activity, because the specimen is collected after a period of long rest and fasting. The first morning urine is more likely to reveal the presence of formed substances and abnormalities. The first morning urine is best for nitrate, proteins, microscopic examination, routine screening and pregnancy test. The random specimen, clean-catch (mid stream) and second morning specimen (double voided) have most common characteristics and minimum bacterial count. These types of specimens are used for chemical screening, routine screening, bacterial culture and microscopic examination. The post prandal and timed 2hr & 12hr urine are used for diabetic monitoring which reflects blood glucose. In these cases total specimen of the time must be collected. In many pathological tests long term 24hr specimen is necessary, because substances excreted by the kidney are not excreted at the same rate or in the same amount during different periods of day and nights. For the measurement of total electrolytes, creatinine, urine protein in diseases more accurate information can be obtained by long term 24hr urine specimen. The urine voided in 24hrs period is collected in to a proper receptacle, a suitable preservative may be added depending on the type of test and collection is refrigerated or in cool chamber, not at higher temperatures. For glucose test, in case of Glucosuria to confirm the diabetes, the specimen need not be refrigerated, but should be kept or stored in a dark bottle and 1gm of boric acid can be added as

6 199 preservative. For ketonic steroids test & ketosteroid to find the adrenal and gonadal abnormalities the specimen should not be refrigerated. However, for normal subject as the rate of excretion of all the constituents substances are within the normal range, a second void can be taken for comparative in vitro studies as the specimen are free from contaminations. The physical properties such as specific gravity, viscosity, surface tension and ph are sex independent. Since the specific gravity of urine is an additive property of its constituents, 80 to 85 percent of USG from normal subjects can be accounted for urea, chloride, sulfate, phosphate and creatinine. Of these, urea contributes up to 35 percent, chloride and phosphate up to 40 percent and creatinine up to 10 percent. The major single constituent is urea which occurs to protein breakdown [Price et al, 1940]. Dilute urine in normal subjects is due to excretion of large amount of water with reduction of specific gravity contributors, the specific gravity pattern remain same. The urine volume increase when the rate of urea excretion is approximately double by administration of urea with intake of high protein diet, where high USG is found [Addis and Watanabe, 1917]. This indicates that the major constituent of USG is urea. It is observed that the refractive index is proportional to USG. Refractive indices determined by refractometer and found a linear relationship between these two independent variable refractive index and USG.

7 200 In urine without dissolved macromolecules the refractometry and hydrometry are the simplest and accurate means of determination of refractive index and specific gravity respectively. The refractive index is proportional to total dissolved solids. The refractometry is superior to hydrometry as a measure of total urinary solids [Rubini and Wolf, 1956]. However, earlier researchers have not reported the increase in refractive index with addition of glucose, urea and albumin. In the present study, it is found that there is an increase of in USG per every gram (1 percent) of solute added and the corresponding increase in refractive index is It is also found that in case of diabetic urine the refractive index is high. The diabetic urine average value is 1.335, while minimum and maximum values are found to be and respectively, which are very high compared to normal urine refractive index In case of chronic kidney disease, the average refractive index value is 1.332, and USG is low. This low value in chronic kidney disease urine is attributed low urea excretion through urine. In the present study, the surface tension was investigated for both normal, normal urine added with glucose, albumin, urea and bilirubin, and also pathological urine from CKD and diabetic mellitus. It is found that the USG of first morning voided is less compared to that of randomly collected, averaging ± The surface tensionmetry can be used in pathology. Hippocrates suggested that the bubbles in urine are associated with kidney disease. The change in surface tension is responsible for bubbles formation in urine. The

8 201 surface tension is more accurate and continuous predictor of protenuria [Charles Daskin, 2000]. The surface tension of biological liquid does not depend on age and sex, but only under some physiological conditions such as menstrual period and various diseases [Charles Daskin, 2000]. In the present study it is found that the surface tension of pathological urines is low compared to normal urines collected from healthy donors. The pathological urine is characterized by high value of urine glucose in case of diabetic mellitus, low urea and high albumin excretion through urine and high blood urea nitrogen in chronic kidney diseases, large amount of bilirubin in Jaundice. The excretion of albumin and bilirubin through urine lowers the surface tension. In the present in vitro investigation of urine, the urine is added with albumin at different concentrations such as 125, 250, 500, 1000 mg/dl and bilirubin in 2.5, 5, 10, 20 and 40 mg/dl. It is found that as the concentration increases the surface tension also increases. During the present study the urine of both CKD and albumin added urine there were large number of bubbles. The surface tension was found by removing the foam bubbles around the capillary tube three minutes after it is placed in the sample for surface tension determination. The surface tension of urine is significantly different when solutes including bile salt excreted through urine and the bile salt concentration is the main determinant by surface tension of urine [Mills C.O, 1998].

9 202 The glomerulus filtrate may have small proteins, and when the glomeruli are damaged large proteins leak in to urinary tract, this condition is called albuminuria or protenuria. The value of albumin in normal urine is up to 7 mg/dl. In case of CKD the excretion of albumin is very high up to 500 mg/dl. The excretion of urine creatinine in CKD is about 30mg/dl, which is very low compared to 1.5gm/dl in normal urine. The urine albumin to creatinine ratio (UACR) in kidney has been found high. According to National Kidney Foundation (NKF), if the UACR is more than 30mg/gm or 0.03 which is abnormally high, then it is a case of CKD, where the GFR is less than 60 ml/min. In case of chronic kidney disease the value of UACR has been found more than 1.60 which is abnormally high compared to normal value around It has also been found that, the specific gravity, refractive index, viscosity and surface tension of urine in Chronic Kidney Disease are low compared that of to normal urine. The decreased value of surface tension can be attributed to the shape of albumin protein molecule. The albumin protein molecules are ellipsoidal in shape, they need time to develop an orientation at air-water inter face, a process that results in surface tension. At higher concentrations of albumin in urine, the molecules have more difficulty in re-orienting to the surface of urine due to greater electrostatic repulsion. The decreased surface tension and specific gravity of urine is an accurate method of finding the kidney decease. This method of detecting kidney disease or protenuria is a quick and

10 203 cheaper method, and it suggests that the glomerulus filtration rate is low. The presence and addition of bilirubin in urine does not increase the specific gravity, refractive index, viscosity and electrical conductivity. The surface tension is decreased with increasing concentration of bilirubin. It is observed that the diseased or pathological urine has less surface tension compared to normal urine. During the study the ph of urine has been found between and averaging ± The addition of glucose and constituents does not affect the ph. The urine ph never increases. In the present study it is found that the first morning urine is concentrated and has more viscosity than the randomly collected urine, and doesn t depend on the age. It is found that the average viscosity ± An in vitro study of urine added with glucose found that as the concentration of glucose increases, the viscosity increases linearly. The glucose in the glomeruli is reabsorbed back in to the blood stream at proximal convoluted tubule (PCT) only when it is below threshold value 185 mg/dl, and the glucose at PCT is more than the threshold value it is excreted through urine. The glucose excreted in urine is a simple sugar. It is called D- glucose or dextrose. In the present investigation it is found that the specific gravity and viscosity of urines of diabetic mellitus subjects are high compared to normal subjects. The high viscosity is due to glucose present in it. The specific gravity and viscosity are the two significant parameters in the diabetic

11 204 mellitus. In the present study a graph plotted between the blood sugar and viscosity is a straight line, indicating that the viscosity increases with concentration of glucose. It is found that the presence of albumin and bilirubin does not affect the viscosity of urine. The viscosity of chronic kidney disease (CKD) urine has less viscosity compared to normal urine, because of low urea, excretion and high blood urea nitrogen (BUN). But the viscosity of Diabetic CKD urine is equal or comes up to that of normal urine, because of glucose excretion through urine. In the present study it is found that the average value of electrical conductivity of urine is 13.16±5.5. It is observed that the first morning urine has high conductivity compared to randomly collect. The urine added with glucose and urea has low conductivity, the electrical conductivity decreases with increasing concentration. The decrease in the conductivity can be attributed to the dilution effect. The increase in the electrical conductivity may be correlated with RBC, calcium oxalate monohydrate crystals, puss cells and uric acid. High values of specific gravity and total dissolved solids urines have large electrical conductivity. Fourier Transform Spectroscopy of Normal and diseased Urine FT-IR spectroscopy has been used by Biophysicist and chemist as a powerful tool to characterize compounds. It has been applied in biology for studying the structure and conformation of molecules like proteins, nucleic acids and lipids. The advances made in instrumentation have paved the way for its utilization in medicine.

12 205 Besides the application of FT-IR for tissue diagnostics, the investigation of body fluids has been gaining importance. The mid IR region is very useful in the identification of disease patterns using the FT-IR spectrum of human sera. Precise quantification of several components such as glucose, total protein, and urea has been achieved using FT-IR spectroscopy. Measurement of the concentration of urea, glucose, protein, bilirubin and creatinine in human urine has been performed using NIR spectroscopy. In literature it is reported that FT-IR can be utilized for identifying disease patters through analysis of bio fluids. Diagnostic applications of IR will provide the physicians an objective aid in the identification of disease state, for which patter recognition approach can be used. The deviation from the normal health condition, which was reflected in the urine, could be sensed by FT-IR techniques with high reliability before acute manifestation of symptoms. In the past decade the infrared spectroscopy has made important contributions to the arena of hematology. The normal physiology and pathological modification have been investigated. By relating subtle alternations in the concentration and structure of macro and micro molecules in urine, the IR spectroscopy has become an ideal complementary tool to diagnose various urological and nephrological disorders. The IR spectrum is like a finger print or signature of the molecular species making up of the sample. The intensities of IR spectra provide quantitative information while the absorption

13 206 positions reveal qualitative characteristics about the nature of chemical bonds, their structure and their molecular environment. One can study the pathology of urine or any other bio-liquid using the FT-IR spectra. The major absorption bands arise from NH, C=0, C- H and X-O bonds found in urea, carbohydrates, proteins, lipids and nucleic acids. The frequency, intensity and width of the particular vibration IR spectral bands are extremely sensitive to the packing constraints, conformational changes and chemical vibrations in lipids, carbohydrates, urea and proteins present in urine. Specifically vibrations that have been previously used in the study of lipids are the CH2 stretching (2850 and 2920 cm -1 ) bending or scissoring ( cm -1 ) and wagging ( cm -1 ) modes, the lipid finger print region containing phosphate and diester stretch modes ( cm -1 ), and the C=0 stretching ( cm -1 ) vibrations. It can be seen that an overall appearance of urine spectrum is dominated by urea, not surprisingly as urea concentration is expected to be much higher than any other urinary constituent in normal urine. The most intense bands for urea are 3400 and peaks between cm -1 and most intense absorption band in proteins is the amide-i peak, which is observed at 1652 cm -1. Amide I is mainly associated both the C=0 and C-N stretching vibration and is also related to the backbone conformation. The next major absorption band is the amide-ii that derives largely from the in plane N-H bending, and the C-N and C-C stretching vibrations.

14 207 It is found that the protein content in the urine is always lower than that in the blood plasma. This agrees with the fact that the major constituent of urine is urea, and the blood cells contain more than 35% hemoglobin. FT-IR method is simple, rapid and effective at probing urine urea, glucose and protein changes. Some preliminary conclusions can be drawn with regard to the interpretation of FT-IR spectra of urine samples. The region between 4000 and 3100 cm -1 is dominated by rather broad spectral features resulting from O_H stretching modes (~3400 cm -1 ) and from N-H stretching modes (amide-a ~3300 cm -1 ). The region between 3100 and 2800 cm -1 exhibits the C_H stretching vibration of CH3 and; CH2 functional groups and, hence, in generally dominated by the fatty acids of the various membrane amphiphiles like phospholipids and by some amino acid side chain vibrations. Complementary information can be deduced from the region between 1470 and 1350 cm -1, where the various deformation modes of these functional groups are located in the spectrum. The region between 1800 and 1500 cm -1 is dominated by the conformation sensitive amide-i and amide-ii bands, which are most intensive bands in the spectra of nearly all complex biological systems so far tested. It is found in the present investigation that a cm -1 is creatinine and the most specific peak is 1493 cm -1 as the value of creatinine is low in normal urine, it cannot be found. But at higher concentrations of creatinine the intensity of the peak at 1490 cm -1 is increased.

15 208 In the present investigation the peak at 1450 cm -1 is observed whose intensity is increasing with a concentration of albumin in urine. It can be seen that the peak at 1450 cm -1 is the most specific peak for albumin in urine. The present investigation s results are in agreement with the earlier researchers. Since IR spectroscopy is an averaging technique, the amide-i and amide-ii bands cannot provide structures information on a single protein but rather they indicate the predominance of structure present. In conclusion, the present study has demonstrated that FT-IR spectrometry is a useful tool for determining concentration of multiple bio molecules in micro samples of urine. The vibrational frequency of a bond is expected to increase when the bond strength increases, and also when the reduced mass of the system decreases. One can predict that C=C and C=O stretching will have higher frequencies than C-C and C-O stretching, respectively. We also expect to find C-H and O-H stretching absorptions at higher frequencies than C-C and C-O stretching. Continuous measurements are often necessary in intensive care units, which can be realized with extra corporal sensing devices. Sensors using IR spectrometry combined with micro dialysis probes or systems that rely on open tissue perfusion. Noninvasive technology is based mainly on near infrared spectroscopy.

16 209 FT-IR spectroscopy has been employed to the past three decades as a research tool in the study of the structure of bio molecules. Ideal techniques for the rapid characterization of microbial pathogens would include those which require minimal sample preparation, permit the automatic analysis of many serial samples with negligible reagent costs, allow their rapid characterization against a stable data base, would be easy to use and would be operated under the control of a PC with recent developments in analytical instrumentation, these requirements are increasingly being fulfilled by pyrolysis mass spectrometry (py MS ) and the vibrational spectroscopic methods of Fourier Transform-Infrared spectroscopy (FT-IR) and dispersive Raman microscopy. The FT-IR spectroscopy can be used to fine the calcium oxalate crystal present in urinary bladder and papillae called renal calculi. The absorption bands due to calcium oxalate are found in the frequency range 1630 cm -1 and 1330 cm -1, due to reduced intensity C=C and O-H bending in plane. FT-IR spectroscopy is an established method for the routine diagnosis of various micro organisms such as yeasts and bacteria on a species level. The cell at large and also its components like nucleic acids, proteins, fatty acids, lipids and polysaccharides can be analyzed by FT-IR spectroscopy. The differentiation of filamentous fungi in the routine laboratory is based on microscopy of the native material addressing morphology and biochemistry of cultures, and to a lesser extent molecular biology.

17 210 FT-IR spectroscopy showed a high reproducibility. The spectral range between 800 and1400 cm -1 turned out to be characteristic of the polysaccharide structure and composition of the capsule of Cryptococcus neoformans. It could be shown that with one frequency range ( cm -1 ) Cryptococcus neoformans var, neoformans separated in two distinct clusters. Biological and biomedical applications of vibrational spectroscopy have witnessed an enormous progress in the past. FT-IR and FT- Raman spectroscopy are used to characterize and differentiate micro organisms, test the susceptibility of pharmaceuticals to bacteria and are to instigate mammalian cells, tissues, body fluids and for infrared imaging. To cross the margin and transfer these elaborated spectroscopic techniques into routine analysis and applications, high requirements in the reliability and simplicity of use are given. The common spectral feature of all complex biological materials is their small spectral variance between the different objects. New strategies for data evaluation are required. Methods of pattern recognition like artificial neural networks with appropriate techniques of spectral feature selection and data processing are promising techniques. For spectral identification, different types of artificial neural network architectures have been applied. With the development of such integrated data elucidation systems, the combination of vibrational spectroscopy with stacked spectra preprocessing, feature selection and artificial neural networks seem to open the perspective

18 211 of a methodology that can be used as an effective tool in diagnosis and therapy.