Author: Dhinaa, A N; Palanisamy, P K
Date published: March 1, 2010
Journal code: JBSN
(ProQuest: ... denotes formulae omitted.)
Protein is an essential nutrient made up of building-block chemicals called amino acids. Protein provides energy and is needed for the body to make new cells, to maintain and rebuild muscles, to carry other nutrients, to act as messengers in the body, and to support the immune system. A total serum protein test measures the total amount of protein in the blood. It also measures the amounts of two major groups of proteins in the blood: albumin and globulin.
Albumin is made mainly in the liver. It helps keep to the blood from leaking out of blood vessels. Albumin also helps to carry some medicines and other substances through the blood and is important for tissue growth and healing.
Globulin is made up of different proteins called alpha, beta, and gamma types. Some globulins are made by the liver, while others are made by the immune system. Certain globulins bind with hemoglobin. Other globulins transport metals such as iron in the blood and help fight infection.
Low total protein levels can suggest a liver disorder, a kidney disorder, or a disorder in which protein is not digested or absorbed properly. Low levels may be seen in severe malnutrition and with conditions that cause malabsorption, such as Celiac disease or inflammatory bowel disease (IBD). High total protein levels may be seen with chronic inflammation or infections such as viral hepatitis or HIV. They may be caused by bone marrow disorders such as multiple myeloma.
Measurements of protein may reflect liver disease, nutritional state, kidney disease and others. A decreased value of total protein may indicate liver or kidney disease. If levels of albumin are low, there is a possibility of primary liver disease, kidney disease, tissue damage or inflammation, and malnutrition [1,2]. In chronic liver diseases like "cirrhosis" or "nephrotic syndrome" the level is decreased. Poor nutrition or protein catabolism may cause "hypoalbuminaemia". Measurement of serum-total protein is useful in conditions relating to changes in plasma or fluid volumes, such as shock and dehydration. In these conditions concentration of serum-total protein is elevated indicating hemoconcentration. Haemodilution is reflected as relative hypoproteinemia, which occurs with water intoxication or salt retention syndrome, during massive intravenous infusions.
The most widely accepted assays so far for proteins are the Biuret , Lowry , Bradford [5,6], Bromophenol Blue  and Bromocresol Green methods. In this Biuret reaction is highly susceptible to interference by non-protein substances [9,10,11,12]. The bromocresol green method for determination of serum albumin is the most specific and sensitive of the dye binding techniques . The glyoxylic acid method measures tryptophan content which represents 8-10% albumin and 90-91% globulin. Since the bromocresol green method is specific and simple, it is the method of choice for albumin determination .
The Z-scan technique was extended to study the optical nonlinearity has been reported for LDL-Cholesterol [15,16]. Some more reports are on characterization of lipids in body fluid [17,18], study of the nonlinear refraction of vitreous humor in human and rabbit , determination of nonlinear refractive index of retinal derivatives . In this present investigation total protein and albumin are subjected to the Z-scan technique to calculate the nonlinear refractive index (n^sub 2^). Already work has been done on measurement of glucose , total cholesterol and triglycerides .
The single beam Z-scan analysis, which was developed by Mansoor Sheik Bahae et al. , is a simple and effective tool for determining nonlinear optical properties of materials [24,25,26,27]. This approach has been now a day widely used in optical characterization of different materials. Nonlinear refractive index is proportional to the real part of the third-order susceptibility Re[χ(3)]. Basically, the Z-scan method consists in translating a non-linear sample through the focal plane of a tightly focused Gaussian laser beam and monitoring the changes in the far field intensity pattern. For a purely refractive nonlinearity, the light field induces an intensity dependent non-linear phase and, as consequence of the transverse Gaussian intensity profile, the sample presents a lens-like behavior. The induced self-phase modulation has the tendency of defocusing or recollimating the incident beam, depending on its Z position with respect to the focal plane. By monitoring the transmittance change through a small circular aperture placed at the far field position, it is possible to determine the nonlinear refractive index. In the present study, we have measured total protein and albumin levels in blood by calculating the nonlinear refractive index (n^sub 2^) value using a single beam Z-scan method.
2.1. Preparation of Total Protein Sample
For sample preparation (Total Protein-Biuret method - a kit supplied by Transasia Bio-medicals Ltd, Baddi, Himachal Pradesh, India) was used. To 20 microliter of the serum one milliliter of total protein reagent was added and incubated for 10 minutes at 37 °C. The principles involved for this reaction is that the peptide bonds of protein react with copper II ions in alkaline solution to form blue-violet complex (Biuret reaction). Each copper ion complexes with 5 or 6 peptide bonds. Tartrate is added as a stabilizer whilst Iodide is used to prevent auto-reduction of the alkaline copper complex. The color formed is proportional to the protein concentration.
2.2. Preparation of Albumin Sample
For sample preparation (Albumin-BCG method - a kit supplied by Transasia Bio-medicals Ltd, Baddi, Himachal Pradesh, India) was used. To 10 microliter of the serum one milliliter of albumin reagent was added and incubated for 1 minute at 37°C. The principle involved in this reaction is that the albumin binds with Bromocresol green (BCG) at pH 4.2 causing a shift in absorbance of the yellow BCG dye. The Blue green color formed is proportional to the concentration of albumin.
2.3. Nonlinear Refractive Index
The Z-scan experiments were performed using a 532 nm Nd: YAG (SHG) CW laser beam (COHERENT-Compass 215M-50 diode-pumped laser) and He-Ne laser beam (RESEARCH ELECTRO OPTICS-30995 cylindrical helium-neon laser) focused by a lens of 35 mm focal length. The experimental set up is shown in Figure 1.
A typical closed-aperture Z-scan curve for the standard total protein solution at incident intensity Io = 7.824 kW/cm^sup 2^. Likewise the Z-scan curve for standard albumin solution at incident intensity Io = 1.758 kW/cm^sup 2^. This normalized transmittance curves are characterized by a prefocal peak followed by a post-focal valley. This implies that the nonlinear refractive indices of total protein, albumins are negative (n^sup 2^ < 0). The defocusing effect shown in Z-scan curve can be attributed to a thermal nonlinearity resulting from absorption of radiation at 532 nm and 633 nm respectively. Localized absorption of a tightly focused beam propagating through an absorbing sample medium produces a spatial distribution of temperature in the sample solution and consequently, a spatial variation of the refractive index, that acts as a thermal lens resulting in phase distortion of the propagating beam.
The nonlinear refractive index (n^sup 2^) is calculated using the standard relations .
Where ΔTp -v can be defined as the difference between the normalized peak and valley transmittances ... is the on-axis phase shift at the focus.
The linear transmittance of the aperture is given by
where r^sub a^ is the radius of the aperture and wa is the beam radius at the aperture.
where n^sub 2^ is the nonlinear refractive index, k is the wave number ... and
... is defined as the peak intensity within the sample at the focus. L is the thickness of the sample, α is the linear absorption coefficient.
An additional experiment was performed with a conventional colorimetric method following the standard procedure of A. G. Gornall et al.  and R. L. Rodkly et al.  for total protein and albumin samples respectively. This involves measurement of optical density variation with respect to concentration. These results are compared with the results calculated with the Z-scan technique.
2.4. Statistical Analysis
The error involved in the measurements was determined by t test, P < 0.01.These statistical analysis was conducted using SPSS commercial statistical package (SPSS, version 10.0 for windows, SPSS Inc., Chicago, U.S.A).
3. RESULTS AND DISCUSSION
3.1. Measurement of Absorbance Spectra
The absorption spectra were measured using UV-Vis spectrophotometer (SHIMADZU-UV-2401PC), and the spectra for both total protein and albumin were found to be broad banded as depicted in Figure 2. Hence for further study 532 nm Nd:YAG laser for total protein and 633 nm He-Ne laser for albumin were used.
3.2. Measurement of Nonlinear Refractive Indices
The results of typical Z-scan normalized transmittance measurement for total protein and albumin are shown in Figure 3. As the concentration of the total protein and albumin increases, the normalized transmittance peak increases whereas the valley decreases respectively. The graph in Figure 4 (a) and (b) shows that the ΔTp-v value linearly increases with concentration of standard total protein and albumin solutions. Similarly in Figure 4 (c) and (d) refractive index value linearly increases with concentration of standard total protein and albumin solutions.
In addition experiment based on optical density is given in Figure 5 (a) and (b). The experiments were repeated five times and the mean value of the nonlinear refractive index (n^sub 2^) was calculated from the normalized transmittance values. This calculated value was assumed to be the standard for measurement of unknown total protein and albumin content present in blood sample. This can be arrived by plotting a linear graph of total protein and albumin concentration Vs nonlinear refractive index. The nonlinear refractive index value was first measured against the reagent blank solution. The calibration was made with the conventional colorimetric method and the results are tabulated in Table 1 for total protein and in Table 3 for albumin. The normal level of total protein in serum is in the range of 6-8.3 g/dl, and serum albumin normal level is in the range of 3.2-5 g/dl.
For estimating the total protein and albumin levels, one need not plot full Z-scan curve every time. Once, experimental setup explained above is established, one needs to note down peak and valley values of the transmittance curve translating the sample holder continuously along Z-axis. The difference in these two values Tp-Tv, |ΔΦ^sub 0^| when substituted in Equation (3) yields the nonlinear refractive index value.
Consequently by the results of Z-scan method, we infer that the n^sub 2^ value is to be in the range of 13.90 ± 1.98 to 23.01 × 10^sup -8^ cm^sup 2^/W for normal level of total protein in serum. Likewise, n^sub 2^ value for normal level of albumin in serum is to be in the range of 5.26 to 8.16 ± 0.98 × 10^sup -8^ cm^sup 2^/W.
3.3. Valuation with Conventional Method
Many trials were performed to measure the total protein and albumin level with Z-scan method. The blood samples were collected from five volunteers. We could see that the results arrived are in good agreement with those of the conventional colorimetric method for total protein as shown in Table 2 and for albumin Table 4. Hence we could clearly ascertain that the Z-scan method is on par with the conventional colorimetric method. By calculating the total protein and albumin values we can also calculate the globulin level in serum. (Globulin = Total Protein-Albumin) is tabulated in Table 5.
The Z-scan measurements indicate that the total protein's and albumin's standard sample and serum sample exhibit nonlinear optical properties. We have measured the nonlinear refractive index values for total protein and albumin present in the serum sample by Z-scan method with 532 nm Nd:YAG CW laser and 633 nm He-Ne laser respectively. Comparative analysis of these values with the one obtained by conventional colorimetric method shows that they are in good agreement. Hence, apart from existing techniques, Z-scan technique can also be used for the measurement bioanalytes in serum.
 Tietz, N.W. (1991) Clinical guide to laboratory tests, 2nd Edition, Saunders Co.
 Friedman, R.B and Young, D.S (1997) Effects of disease on clinical laboratory tests, 3rd Edition, AACC Press, Washington, DC.
 Gornall, A.G., Bardawill, C.J. and David, M.N. (1949) Determination of serum proteins by means of the Biüret reaction. The Journal of Biological Chemistry, 177, 751-766.
 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folinphenol reagent, Journal of Biological Chemistry, 193, 265-275.
 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry, 72, 248-254.
 Zor, T. and Selinger, Z. (1996) Linearization of the bradford protein assay increases its sensitivity: Theoretical and experimental studies. Analytical Biochemistry, 236, 302-308.
 Flores, R. (1978) A rapid and reproducible assay for quantitative estimation of protein using bromophenol blue. Analytical Biochemistry, 88, 605-611.
 Lee Rodkly, F. (1964) Binding of bromocresol green by human serum albumin. Archives of Biochemistry and Biophysics, 108, 510-513.
 Caraway, W.T. and Kammeyer, C.W. (1972) Chemical interference by drugs and other substances with clinical laboratory test procedures. Clinica Chimica Acta, 41, 395-434.
 Elking, M.P. and Kabat, H.F. (1968) Drug induced modifications of laboratory test values. American Journal of Hospital Pharmacy, 25, 485-519.
 Parvin, R., Pande, S.V. and Venkitasubramanian, T.A. (1965) On the colorimetric biuret method of protein determination. Analytical Biochemistry, 12, 219-229.
 De Ia Huerga, J., Smetters, G.W. and Sherrick, J.C. (1964) Colorimetric determination of serum proteins: The biuret reaction. In: Sunderman, F.W., Jr., Eds., Serum Proteins and the Dysproteinemias, Lippincott, Philadelphia, 52-62.
 Doumas, B.T. and Biggs, H.G. (1972) Standard Methods of Clinical Chemistry, Academic Press, New York, 7.
 Doumas, B.T., Watson, W.A. and Biggs, H.G. (1971) Albumin standards and the measurement of serum albumin withbromocresol green. Clinica Chimica Acta, 31, 87-96.
 Kroll, M.H. and Chesler, R. (1998) The nonlinearity seen for ldl-cholesterol with lyophilized material is a matrix effect. Clinical Chemistry, 44, 1770-1771.
 Kroll, M.H. and Chesler, R. (1994) Nonlinearity of high-density lipoprotein cholesterol determinations is matrix dependent. Clinical Chemistry, 40, 389-394.
 Gomez, S.L., Turchiello, R.F., Juradoc, M.C., Boschcov, P., Gidlund, M. and Figueiredo Neto, A.M. (2004) Characterization of native and oxidized human low-density Physics of Lipids, 132, 185-195.
 G'omez, S.L., Turchiello, R.F., Juradoc, M.C., Boschcov, P, Gidlund, M. and Figueiredo Neto, A.M. (2006) Thermallens effect of low density lipoprotein lyotropic-like aggregates investigated by using the Z-scan technique. Liquid Crystal Today, 15, 1-3.
 Rockwell, B.A., Roach, W.P., Rogers, M.E., Mayo, M.W., Toth, C.A., Cain, C.P. and Noojin, G.D. (1993) Nonlinear refraction in vitreous humor. Optics Letter, 18, 1792- 1794.
 Bezerra, A.G., Jr., Gomes, A.S.L., de Melo, C.P. and de Arafijo, C.B. (1997) Z-scan measurements of the nonlinear refraction in retinal derivatives. Chemical Physics Letters, 276, 445-449.
 Dhinaa, A.N., Ahmad, Y.N., Murali, K. and Palanisamy, P.K. (2008) Z-Scan Technique as a Tool for the Measurement of Blood Glucose. Laser Physics, 8, 1212-1216.
 Dhinaa, A.N. and Palanisamy, P.K. (2009) Z-scan technique for measurement of total cholesterol and triglycerides in blood. Journal of Innovative Optical Health Sciences, 2, 295-301.
 Sheik Bahae, M., Said, A.A., Wei, T.H., Hagan D.J. and Vanstryland, E.W. (1990) Sensitive measurement of optical nonlinearities using a single beam. Quantum Electron, 26, 760-769.
 Qusay, M.A. and Palanisamy, P.K. (2005) Investigation of nonlinear optical properties of organic dye by Z-scan technique using He-Ne laser. Optik, 116, 515-520.
 Madhanasundari, R. and Palanisamy, P.K. (2006) Optical nonlinearity of a triphenyl methane dye as studied by Z-scan and self-diffraction techniques. Modern Physics Letter B, 20, 887-897.
 Qusay, M.A. and Palanisamy, P.K. (2006) Z-scan determination of the third order optical nonlinearity of organic dye nileblue chloride. Modern Physics Letter B, 20, 623-632.
 Dhinaa, A.N., Ahmad, Y.N. and Palanisamy, P.K. (2007) Nonlinear optical properties of acid orange 10 dye by Z-scan technique using Ar+ laser. Journal of nonlinear Optical Physics and Materials, 16, 359-366.
Department of Physics, Anna University Chennai, Chennai, India.
Received 19 December 2009; revised 28 December 2009; accepted 4 January 2010.