Total protein is the sum concentration of all individual serum proteins (g/dL). There are many hundreds of different protein species in serum, including straight polypeptides as well as glycosylated and lipid-associated forms. Measurement of total protein for clinical purposes commonly is performed by the following methods.
Methods used to measure total protein
Refractometry
This method is used for estimating plasma protein (including fibrinogen) in EDTA plasma and is reported on routine hemograms at Cornell University. It measures the refractive index of a sample relative to the refractive index of water. The reading is actually a measurement of total solids and is only an estimate of protein concentration, since variation in other serum components “solids” (sodium, chloride, phosphate, glucose, cholesterol, urea, etc.) also can affect refractive index, as can colloids such as hetastarch and gelatin (Yam et al 2018). Lipemia falsely increases the results and moderate to severe hemolysis can blur the refractometer line, making it difficult to read, but bilirubin does not interfere with results (Gupta and Stockham 2014). For more on estimation of total protein with refractometry, refer to the hemogram basics section of the website. There are also different refractometers available, which may not give equivalent results, depending on whether they use a correction formula or not.
Biuret Method
This is the colorimetric method used on the automated chemistry analyzer. It detects all proteins and is accurate for the range of 1-10 g/dL. It is not sensitive enough for low concentrations found in some body fluids such as CSF, urine, and many body cavity effusions.
Note that the total protein (as measured by refractometer) provided with the hemogram is usually higher than that provided from the chemistry analyzer, this is particularly in dogs and cats versus large animal species (in our experience). This difference is attributed to:
- The contribution of total solids to the refractive index.
- The contribution of fibrinogen to total protein content in plasma compared to serum. Note that if the chemistry panel is performed on heparinized plasma, the difference between the two measurements is much less as fibrinogen is present in both EDTA and heparinized plasma samples.
However, it should be noted that there are some reports where the total protein by biuret is higher (0.5-0.7 g/dL on average) than that by refractometry in ruminants, for unclear reasons (Katsoulos et al 2017).
Turbidometric methods
Quantitation of protein in cerebrospinal fluid, urine and other low-protein fluids requires more sensitive techniques than either the Biuret or refractometer method. Protein in these fluids can be measured more accurately with precipitation or dye-binding methods. Precipitation methods include trichloroacetic acid and sulfosalicylic acid, whereas dye-binding uses dyes such as Coomassie blue and pyrogallol red-molybdate. The procedure on the Hitachi 911 at Cornell University for measurement of protein in low-protein fluids is a turbidometric method based on precipitation of protein by benzethonium chloride. It is sensitive to as little as 6 mg/dL of protein.
Note that urinary dipsticks can be used to estimate protein in cerebrospinal fluid samples, however they are not as accurate as turbidometric techniques.
Physiology
Plasma proteins are a very heterogenous group of over a thousand individual proteins, with a variety of functions including maintenance of oncotic pressure, transportation of substances, coagulation, etc. Total protein is often divided into two categories, albumin and globulins. Albumin is a single protein that accounts for about half of the total protein concentration in plasma. The remaining proteins are collectively termed globulins.
Methodology
Reaction type
End-point (bichromatic)
Procedure
- Colorimetric assay (Biuret): In an alkaline medium, divalent copper reacts with protein peptide bonds forming a purple-colored biuret complex that is measured photometrically. The color intensity generated by this complex is directly proportional to the total protein concentration.
Reaction shown below:
Protein + Cu2+ alkaline solution > Cu-protein complex
Units of measurement
Total protein concentration is measure in g/dL (conventional units) and g/L (SI units). The conversion formula is shown below:
g/dL x 10 = g/L
Sample considerations
Sample type
Serum and plasma. Serum and plasma should be separated from the clot within 4 hours of collection.
Anticoagulant
Heparin or EDTA
Stability
The stability of total protein in human serum and plasma samples are as follows: 3 days at 2-8 °C or 6 months at (-15) to (-25) °C.
Interferences
- Lipemia: Severe lipemia (>1000 lipemia index) will increase results.
- Hemolysis: Will increase with severe hemolysis (>650 hemolysis index), as hemoglobin will react as a protein in the assay
- Icterus: Severe icterus may decrease concentrations (>21 icteric index, for the Roche P Modular analyzer). The total protein by biuret was falsely decreased in a dog with severe icterus secondary to immune-mediated hemolytic anemia, whereas the refractometer measurement of total protein was unchanged (Garner et al 2014). A subsequent study with the Roche 6000 C501 module showed that total protein concentration was falsely decreased with increasing concentrations of unconjugated bilirubin (serum samples spiked with bilirubin). In one set of spiked samples, the total protein concentration were affected (decreased by 0.2 g/dL) at 10.7 mg/dL bilirubin (icteric index of 16) and linearly decreased from 5.2 g/dL to 4.1 g/dL at 43 mg/dL bilirubin. In a second set of spiked samples, a decrease in total protein began at 20.8 mg/dL bilirubin (next lowest tested bilirubin concentration was 10 mg/dL) with a similar degree of decrease in total protein (1.0 g/dL) at the highest bilirubin concentration (36 mg/dL). As for the case report, protein concentrations measured by refractometer were unchanged (Gupta and Stockham 2014).
- Drugs: 4% succinylated gelatin will falsely increase results (Yam et al 2018).
Test interpretation
Since total protein consists of a composite of albumin and globulins, the result is not interpreted in isolation, but rather is interpreted in context of the changes in albumin and globulins (independently and in relation to each other). Albumin generally accounts for about half of the total protein concentration in plasma.
