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PII: S0263-2241(17)30510-9
DOI: http://dx.doi.org/10.1016/j.measurement.2017.08.013
Reference: MEASUR 4911
Please cite this article as: K. Zheng, L. Wu, Z. He, B. Yang, Y. Yang, Measurement of the total protein in serum by
biuret method with uncertainty evaluation, Measurement (2017), doi: http://dx.doi.org/10.1016/j.measurement.
2017.08.013
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Measurement of the total protein in serum by biuret method with
uncertainty evaluation
1
Beijing University of Chemical Technology, Beijing, China
2
National Institute of Metrology, Beijing, China
*Authors for correspondence
E-mail: yangyi@mail.buct.edu.cn
Address: No. 15 North Third Ring Road, Chaoyang District, Beijing, China
Abstract
As recommended by IFCC, the biuret method is generally used to determine the total protein as a
reference method. In order to achieve accurate measurement of the total protein by biuret method, a
detailed description of establishment, validation and uncertainty evaluation of this method is proposed
in the metrological view in this paper. Then the method was used to determine the total protein in
RELA serum samples and the results of 2015-RELA sample A and B were 73.9 g·L-1 and 58.9 g·L-1,
with the RSD of 1.18% and 1.07%, respectively. The uncertainty was evaluated in detail fully
following the Guide to the Expression of Uncertainty in Measurement, and the expanded uncertainty of
RELA sample A and B were 2.0 and 1.2 g·L-1, respectively. The results have good agreement with
those from other research institutions. The proposed procedure gives a good example to realize an
accurate reference method with strict metrological control, which can benefit not only total protein
measurement but also other biological measurement by reference methods.
Keywords
Biuret Method, Total Protein, RELA-Samples, Uncertainty
1. Introduction
The total protein content in human tissue fluid plays an important role on the state of health [1]. Serum
is the most important tissue fluid in human body, and total serum protein have a variety of functions,
such as maintaining the osmotic pressure of the blood vessel, maintaining the pH of plasma,
transporting various metabolites, regulating the physiological function of the transport material, and
has a close relationship with the immune function of the body, which can give us useful information
about our health condition. The level of the total serum protein reflects the loss of protein caused by
liver function and renal disease. Measurement of total serum protein can be used to monitor the
nutritional status of the body indirectly, and benefit diagnostic of certain diseases [2]. With the reasons
above, the total serum protein is a routine examination in clinical diagnostics.
Numerous measurement methods of total serum protein have been developed because of its importance.
Several methods, such as the Kjeldahl method [3], the biuret method [4, 5], the Lowry method [6], the
Bradford method [7] and the UV absorbance method [8] are widely used. Although the Kjeldahl
method is widely used, it is too tedious and time-consuming to be used as a routine assay procedure. The
Bradford method is easy to be operated with fewer reagents, but it cannot be used for different protein
determination because of the large deviation results. The Lowry method has a high sensitivity, but it is
subject to several interferences originating from the sample matrix and need to control the operation
time accurately. The UV absorbance method is simple to be used, because the total protein in serum
and other proteins solutions can be determinate directly by UV spectrometry and the method allows
almost complete sample reuse after the measurement. For all that, the UV absorbance method has
obvious weakness, because nucleic acids have almost the same absorption wavelength as protein,
which can disturb the protein measurement. Furthermore, the UV absorbance method has lower
sensitivity and its application is limited.
Compared with the above methods, the biuret method has the advantages of easy operation, excellent
precision and less interference. The principle of this method is that the peptide bond of amino acid
residues and two copper ions (Cu2+) can form a purple complex in alkaline condition, and the darkness
of the color is proportional to the content of protein in the sample. When it is compared with the
standard, treated in the same process, such as SRM 927, the protein content can be obtained. The
darkness of the color is only proportional to the number of peptide bond sand it has no obvious
relationship with the type of the protein composition, molecular weight and amino acid residue.
Although the sensitivity of the method is not good and the detection of limit is generally 0.2 ~ 1.7 g·L-1,
it is enough for the measurement of total protein in serum, whose concentration is 60 ~ 80 g·L-1 in
general. Therefore, the biuret method becomes the most popular routine method for the determination
of the total protein in serum, and it was also proposed as a candidate reference method for total serum
protein measurement to establish the traceability chain in clinical diagnostics [9, 10].
In order to measure the total serum protein accurately by the reference method, the method parameters
should be accurately and strictly followed. “Measurement traceability” is widely used by metrologies,
which refers to an unbroken chain of comparisons of any measurement to a known standard.
Calibration with a traceable standard can determine the bias and precision of specific measurement and
ensure the measurement accuracy. In order to ensure the accuracy of total serum protein measurement
in this research, both the physical parameters and the chemical parameters were well calibrated with
known traceable measurement standards or certified reference materials in advance. Then the method
performance was evaluated and applied in measurement of RELA samples. Good results were achieved
according to the feedback. It can be looked as a successful example of realization of an accurate
reference method with metrological techniques.
2.2 Instruments
UV-VIS spectrophotometer: PE, Lambda 950. Volumetric flask: 1L. Pipettes (200 µL, 1 mL): Gilson,
France. Balance: ME235S, 0.01 mg, German Satorius Company. Constant temperature incubation
apparatus: MTC-100, Hangzhou MIULAB Instrument Co. LTD.
where,
cs is the total protein in the sample;
A1s, A2s, A3s and A4s are the absorbance values of samples;
A1std, A2std, A3std and A4std are absorbance values of CRMs;
cstd is the total protein in SRM 927.
(2)
Where,
Cmeasurement is the average of data for one day;
CCRM is the standard value for CRM GBW09187 or BW3627-1.
For the repeatability, two CRMs (GBW09187, BW3627-1) were measured for nine times in one day.
And the data were brought into Formula (3) to calculate the relative standard deviation (RSD). To
investigate the reproducibility of the method, two CRMs (GBW09187, BW3627-1) were analyzed. For
each CRM, three sub-samples were taken and each sub-sample was measured for three times. The same
measurement procedure was repeated for three separated days. And the averages for each sub-sample
were brought into Formula (3) to calculate the RSD.
(3)
In order to calculate the LOD or LOQ, the maximum noise of the absorbance measurement had been
measured. Then, A4s-A3s-(A2s-A1s) in Formula (1) was replaced by 3 or 10 times of the maximum noise
to calculate the LOD or LOQ, respectively.
Based on the mathematical measurement model shown in Formula (1), there were nine variables and
the typical value of each variable was shown in Table 4.
In order to calculate the uncertainty components, the sensitivity factor of each variable was calculated
firstly, which was shown in the following:
cstd
c A4s c A1s 182.99
A4std A3std A2std A1std
cstd
c A3s c A2s 182.99
A4std A3std A2std A1std
( A4s A3s A2s A1s )cstd
c A4std c A1std 156.09
( A4std A3std A2std A1std ) 2
( A4s A3s A2s A1s )cstd
c A3std c A2std 156.09
( A4std A3std A2std A1std ) 2
A4s A3s A2s A1s
ccstd 0.85299
A4std A3std A2std A1std
(1) Uncertainty from the chromogenic and the whole method variation
It is hard to evaluate the uncertainty component from the chromogenic reaction and the whole method
variation by type-B uncertainty evaluation method. Therefore, the type-A method was used and the
standard deviation of RELA sample A measurement was used to calculate the uncertainty component.
s 1.13
um 0.38 g L1 (4)
9 3
Based on the mathematical model and sensitivity factor, the uncertainty component contributed by
protein CRM was calculated as following:
c
us ustd ccstd ustd 0.085 g L1
cstd
The same spectrometer was used to measure all the absorbance in this study, therefore, strong
correlation exists among the variables and correlation factor of 1 was assumed. Based on Formula (1),
the uncertainty came from the absorbance measurement was calculated as following:
0.91 g L1
U =2 uc 2.0 g L1
Therefore, the result of RELA sample A can be expressed as (73.9 ± 2.0) g·L-1. Similarly, the
uncertainty of RELA sample B was evaluated and the measurement result can be express as (58.9 ±1.2
g·L-1).
3.6 Feedback
The feedback from the RELA organizer was shown in Fig.1 and the lab-code 092 indicated our results.
Our results were overlapped with other results from 13 labs among total 17 participants, demonstrating
the accuracy and reliability of our results.
Fig.1 Results of total serum protein measurement (RELA 2015)
4. Conclusion
In this study, accurate and comparable measurement of total serum protein was achieved by fully
following the reference method via metrological techniques. This is a good example to use the
metrology to achieve accurate and comparable measurement. All the apparatus in the experiment were
calibrated and verified carefully with traceable metrological standards and the method performance
was evaluated with CRMs thoroughly. Therefore, the parameters in the reference method can be
followed strictly to ensure the accuracy and comparability of the measurement. Only a small number of
biological measurements can be traced to SI unit and there are still numerous biological measurements
are defined and traced to reference methods nowadays. Therefore, it is can be expanded to realize
accurate and comparable biological measurements according to other reference methods.
Acknowledgement
This work was supported by National Key Scientific Apparatus Development Project
(2012YQ09019708).
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Highlights
· A biuret method to measure the total protein in serum.
· Instruments were certificated and the data used in calculation of uncertainty.
· Two known concentration standard protein to evaluate the method performance.
· Two serum samples given by RELA were measured.
· The calculation of the uncertainty here developed is detailed.
Graphical abstract