determination of total urinary protein, combining lowry sensitivity
TRANSCRIPT
Determination of Total Urinary Protein, Combining
Lowry Sensitivity and Biuret Specificity
Karl Doetsch and Richard H. Gadsden
A highly sensitive method with specificity for the peptidechain backbone was developed fordetermi- nation of total urinary protein. Interfering substances are removed by gelfiltrationand cupricionsare sto- ichiometricallybound to the peptidebonds of protein by the biuretreaction.Ion-exchange characteristics of the gel are neutralized, allowing protein-copper complex to be separatedfrom excess cupricionsby a second gelfiltrationstep.Copper bound to peptide bonds is colorimetrically determined by use of sodi- um diethyldithiocarbamate. Nonprotein substances do not interfereunless they simultaneouslybind to proteinand chelatecopper.As littleas 1 mg of total protein per deciliter can be determined in heteroge- neous biological fluids such as urine.
Additional Keyphrases: normal range #{149}peptide-bound Cu determined with Na diethyldithiocarbamate #{149}inter- method comparison
The diverse physical and chemical properties of the various kinds of human protein have resulted in a large number of analytical methods (1). Each method is affected by external interferences and by inherent biases. Because numerous and often unde- fined substances are present in biologic fluids, ana-
lytical conditions are not always well controlled. Re- sults obtained depend on the method rather than the actual protein concentration. Albumin is a simple protein comprised essentially of only amino acids. Many other protein species contain various amounts of carbohydrates, lipids, and other constituents, and these nonpeptide constituents often contribute non- specifically and erroneously to protein analysis. Some reactions have been considered specific for protein because of the high concentration of protein and the minimum concentrations of interferences present in serum. For example, the biuret reaction is highly susceptible to interference by nonprotein sub- stances (2-5). Besides pigments, any substance con- taining nitrogen, oxygen, or sulfur that is able to
From the Departments of Biochemistry and Clinical Pathology, Medical University of South Carolina, Charleston, S. C. 29401.
Received July 10,1973;accepted Aug. 13, 1973.
complex cupric ion may cause falsely elevated re- sults. The Lowry method has been shown to be even more susceptible to interferences (6-8). Besides pig- ments, any substance able to reduce the Folin phe- nol reagent may cause falsely elevated results.
A suitable method combining both sensitivity and specificity has been lacking for the determination of low concentrations of protein in biologic fluids. Pe- ters (9) proposed that the method of choice for total protein assay in clinical laboratories should be a ver- sion of the biuret reaction, the measurement to be made relative to that for pure bovine serum albumin (or human serum albumin) that previously had been standardized by Kjeldahl analysis. A modification of the biurt method comparable to the Lowry method
in sensitivity was originally described by Nielsen (10) and modified by Westley and Lambeth (11).
Klungsoyr (12) further modified the earlier methods by using gel filtration to remove excess copper from the biuret reaction mixture. None of these three methods, however, was adapted to clinical laborato- ry use.
A semi-automated biuret method for determina- tion of total urinary protein, in which gel filtration is used to remove interfering substances, has been de- scribed by Doetsch (13). The limit of sensitivity is 50 mg/dl total protein in the sample. In the proposed method, removal of unbound copper by an addition- al gel filtration step and subsequent colorimetric assay for copper bound to peptide bonds extends the limit of sensitivity to 1 mg of total protein per decili- ter of sample. Nonprotein substances do not interfere because only the peptide chain backbone (-RCH- CO-NH-) contributes to the analysis. The new and highly sensitive modified biuret assay described here is a true determination of protein peptide back- bone as suggested by Nielsen (10) for a standard ref- erence method.
Materials and Methods
60 g 40 g 15 g
5g 1 liter
2 Columns are prepared by cutting off the tip and reaming the
opening with blunt-end surgical scissors. The enlarged opening assures a more rapid and uniform flow. Columns and funnels are washed with a test tube brush and tap water at the end of the day and can be re-used indefinitely.
Biuret reagent should not be added to the collection cups be- fore the column effluent has been collected completely, because protein sometimes coagulates as a result of the initial high rela- tive base concentration that exists as the first few drops are col- lected.
We have found that distilled water may be used as the eluent in the second gel filtration instead of the sodium hydroxide re- agent.
Manual colorimetry can be performed by adding 2.0 ml of DTC color reagent to the second column effluents and measuring the color developed after 10 minutes but within one hour.
CLINICAL CHEMISTRY, Vol. 19, No. 10,1973 1171
oratories, San Mateo, Calif. 94401): Plastic column
chromatography racks and trays, Cat. No. 0904. Polyethylene column elements, 11 mm in diameter and 50 mm high, Cat. No. 0956. Polyethylene col- umn funnels, Cat. No. 0901.
Single-channel “AutoAnalyzer” system (Techni- con Corp., Tarrytown, N. Y. 10591).
Reagents
Only analytical reagent-grade chemicals and dis- tilled water were used.
Sephadex G-50-80 (Pharmacia Fine Chemicals, Piscataway, N. J. 08854). The gel was hydrated ac- cording to manufacturer’s directions (14). A large- bore, 10-ml serological pipette was used to charge the columns.
Biuret reagent. A modification of Weichselbaum’s reagent (15) was prepared. Final sodium hydroxide concentration was 0.2 mol/liter. Additional sodium potassium tartrate was used to prevent turbidity in the reaction mixture.
NaKC4H4O6.4H20 NaOH CuSO4.5H2O KI Distilled water, dilute to
Sodium diet hyldithiocarbamate color reagent. Two grams of the crystalline salt (Mallinckrodt Chemical Works, Jersey City, N. J. 07303) was dis- solved in 1 liter of distilled water. This was a 20-fold excess over minimum concentrations required for lin- earity. The reagent is stable indefinitely at room temperature. Five-tenths milliliter of “Brij-35” sur- factant (Technicon) was added per liter for continu- ous-flow colorimetry.
Sodium hydroxide reagent, 0.1 mol/liter. Human serum albumin (Dade Division, American
Hospital Supply Corp., Miami, Fla. 33152). Purity and char’acter of Lot No. PRS-410 were verified by ultracentrifugal analysis, cellulose-acetate electro- phoresis, and spectrophotometric absorption analysis according to the criteria of Peters (9). Total protein value of 7.8 g/dl was assigned by micro-Kjeldahl ni- trogen analysis (nitrogen value multiplied by the factor 6.25). HSA1 was determined to be more than 99.5% pure and to contain less than 5% aggregates. Working standards in the range 2-80 mg/dl were made by dilution with physiological saline. These standards are stable for about a month stored at 25 #{176}Cin brown bottles if a few crystals of sodium azide are added.
A bnormal urine chemistry control (Lederle Diag- nostics, Pearl River, N. Y. 10965), Lot No. 2921- 695H1, was diluted two-fold with physiological saline and stored as were the working standards.
1 Nonstandard abbreviations used: HSA, human serum al- bumin (Dade Division, American Hospital Supply Corp., Miami, Fla. 33152); DTC, sodium diethyldithiocarbamate; HSP, human serum protein reference standard (4.0 g of albumin and 3.5 g of y-globulin per deciliter; lot No. 2C4V; Dow Diagnostics, India- napolis, hid. 46206); TCA, trichloroacetic acid.
Procedure
Preparation of columns:2 A set of two columns is used for each standard, sample, and control. For ini- tial gel filtration, charge the columns with Sephadex
G-50-80 until the top of the gel is approximately 2 mm from the top of the column. For the second gel filtration, charge the columns with Sephadex G-50- 80 until the top of the gel is about 4 mm from the top of the column. (The gel expands when base is added.) Insert polyethylene funnels into the col- umns. Add 15 ml of sodium hydroxide reagent to each column to be used for copper removal in the second gel filtration. After the sodium hydroxide re- agent has passed through, this column is ready for removal of excess copper, because the weak ion-ex- change properties of the gel have been neutralized, thus preventing loss of copper on the gel. All col- umns are prepared freshly each day, but may then be used throughout the working day.
When a diluted aliquot of biuret reagent (contain- ing no protein) was treated by the copper removal step, the absorbance reading of the effluent to which
DTC reagent had been added was zero, indicating that excess copper is completely removed and that there is no copper in the column bleed.
Gel filtration: Apply 1.0 ml of working standards, samples, and controls to individual columns. Allow the liquid to percolate into the gel until column flow ceases. Remove the last hanging drop. Insert a 5-mi polystyrene AutoAnalyzer cup into the rack beneath the column and then gently apply 2.0 ml of distilled water to the top of each column. Collect the effluent until column flow ceases, including the last hanging drop. Add 0.5 ml of biuret reagent to each cup,3 mix well (mixing is conveniently accomplished by gently injecting air bubbles with a disposable Pasteur pi- pette), and allow to stand at room temperature. After 30 mm, apply 1.0 ml of the biuret reaction mixture to a base-conditioned column and allow to percolate into the gel until column flow ceases. Re- move the last hanging drop. Place a 5-mi polystyrene cup beneath each column and then carefully apply 2.0 ml of sodium hydroxide reagent4 to the top of each column. Collect the effluent until column flow ceases, including the last hanging drop.
Colorimetry: The second gel effluent may be ana- lyzed by manual5 or automated colorimetry. Figure 1
EwufNTII.
Fig. 3a. Elution profile (Sephadex G-50-80), first gel fil- tration
1.0
0.8
0.6
0.4
30/H
SAMPLER
To Sample, Wash Receptacle
Waste - p .056 FROM F/C
Fig. 1. Diagram of manifold for determination of total uri- nary protein by the biuret:DTC method
Fig. 2. Strip-chart recording, illustrating linearity, carry- over, and steady state
shows the manifold diagram. The working standards, samples, and controls are analyzed for copper by using the sodium diethyldithiocarbamate color re- agent (16). The yellowish-brown color is measured at 440 nm. Ninety-seven percent of full color develop- ment occurs within 3 mm, but the color must be measured within 1 h because the copper diethyldi- thiocarbamate complex is unstable. Decreasing per- cent transmittances of working standards are plotted vs. HSA concentrations. Sample and control values are read directly from the chart.6
Results Evaluation of Continuous-Flow Colorimetry
Variation of continuous-flow colorimetry for sam- ples run successively was less than 1%. Precision for
samples run in mixed orders and interaction between successive samples were acceptable. Figure 2 is a typical strip chart recording illustrating the standard curve, sample interaction, and steady state. Note that sample interaction was reduced by using DTC color reagent in place of distilled water in the Sam- pler II wash receptacle.
Gel Filtration and Linearity
The eiution profile for initial gel filtration (Figure 3a) was determined in fractions from the column by
6 We have found that the series of working standards can be
prepared in advance by individually pooling the gel effluents from several columns. These may be stored in brown bottles at room temperature and used to calibrate each run. A control, however, is included with each group of samples.
Fig. 3b. Elution filtration
use of the biuret reaction and a “Chloride Meter 920” (Corning Glass Works, Sci. Instruments Div., Medfield, Mass. 02052). At least 92% of salts and
small molecules are removed from urine by the first gel filtration step. The protein elution profile for the second gel filtration (Figure 3b) was determined by using DTC color reagent with column fractions.
Recovery from gel filtration of protein from solu- tions of various concentrations of HSA, HSP, and clinical urine samples is constant for different pro- tein species tested (Table 1), but reflects the phe- nomenon of less-efficient gel filtration behavior of very dilute protein solutions as compared to more concentrated ones. Recovery of protein from the first gel filtration step was determined by applying 1.0 ml
Added Determined Recovery
Added Determined Added Determined Recovery Recovery
mg/mi % mg/mi %
0.085 87 0.185 95 0.366 94 0.539 93 0.590 0.560 95 0.755 97 0.740 0.710 95 0.947 97 1.91 98 1.85 1.74 94 2.56 98 2.47 2.38 97 3.86 99 3.70 3.65 99 7.74 99 7.40 7.28 98
0.016 66 0.037 0.024 68 0.032 65 0.074 0.049 66 0.064 66 0.150 0.103 68 0.130 67 0.220 0.151 69 0.255 65 0.370 0.261 70 0.414 71 0.590 0.446 76 0.563 72 0.740 0.555 75
1.32 1.31
2.75 2.72
0.045 0.030
0.300 0.020
99
99
67
67
O Results of triplicate determinations. with use of Sephadex G-50.80. Human serum albumin (Dade Division). Human serum protein (Dow Diagnostics): 4.0 g albumin and 3.5 g gamma globulin per deciliter.
10
Fig. 4a. Standard curve for HSA, absorbance vs. concen- tration
Table 1. Protein Recovery from Gel Filtration’
CLINICAL CHEMISTRY. Vol. 19, No.10,1973 1173
mg/mi
2.60
3.90
7.80
0.049
0.098 0.195 0.390 0.585 0.780
of protein solution to a gel column and eluting with 2.0 ml of distilled water. A second 1.0-ml aliquot was
diluted to 2.0 ml with distilled water. Biuret reagent was added to both sets and, after 30 mm, absorban- ces were measured at 550 nm. Recovery of protein from the second gel filtration was determined by isolating the biuret protein-copper complex and sub-
sequently applying 1.0 ml of the ensuing protein- copper solution to a base-treated gel column. The el- uent used was 2.0 ml of sodium hydroxide reagent. A second 1.0-ml aliquot was diluted to 2.0 ml with so- dium hydroxide reagent. DTC color reagent was added to both sets and, after 10 mm, absorbances were measured at 440 nm.
A representative standard curve for HSA (absorb- ance) is illustrated in Figure 4a. The lag at low con- centrations results from the aforementioned gel-fil- tration behavior. The relationship between absorb- ance and protein concentration is essentially linear from 20 to 80 mg/dl. The lag problem in standard- ization is circumvented by the use of an AutoAnalyz- er chart reader whereby percent transmittance is plotted vs. HSA concentration (Figure 4b). By this technique, a straight line is obtained from 2 mg/dl to 20 mg/dl, the portion of the curve where sensitivi- ty is of greatest importance.
Precision
The largest coefficient of variation for within-run precision was 5%, and precision increased with pro- tein concentration (Table 2). Mean ± SD for three dilutions of the “Abnormal Urine Chemistry Con- trol” assayed once daily over 20 days was 16 ± 1, 33
± 2, and 67 ± 3 mg/dl. Best results were obtained by using glass pipettes.
Accuracy
Known amounts of HSA were added to previously
analyzed clinical urine samples. The results of recov- ery studies are presented in Table 3. Starting con- centrations of three clinical urine samples and of HSA solution added were determined by taking the mean value of 10 analyses. Various amounts of HSA solution were added to 100-ml aliquots of each urine sample, and the resulting samples then were ana- lyzed in triplicate. Recovery of added HSA was 101 ± 3%.
‘C
a
Protein concen- tration mg/dl
Fig. 4b. Standard curve for HSA, percent transmittance vs. concentration
B. Clinical urine samples
Normal Values
0.212 ± 0.002 0.426 ± 0.005
0.642 ± 0.006 0.850 ± 0.002
0.068 ± 0.001 0.107 ± 0.003 0.147 ± 0.001 0.293 ± 0.009 0.684 ± 0.009 0.934 ± 0.19
#{176}Results of quintuplicate determinations.
Coefficient of variation
5
2
3
3
3
2
Urinary 24-h protein excretion for 88 healthy white adults was determined over an eight-week period by
the biuret:DTC method.7 The range of total creati- nine excretion was 1.12 to 2.52 g per day. Subjects were volunteers from the student body and staff at the Medical University of South Carolina. Eighty were men between 22 and 36 years of age; three subjects were older than 36 years but younger than
55 years. Five subjects were women 22 to 29 years old. The values for all the women fell below the mean value, but there was no grossly discernible bias
because of age or sex. The medical histories of the subjects were screened by personal interview, ques- tionnaires, and examination of student health rec- ords. Consumption of all medications and of alcohol was controlled by requesting abstinence for 48 h be- fore and during the collection period. Small amounts of common medications (antihistamines, aspirin, oral contraceptives) taken by four subjects and small amounts of alcohol consumed by eight subjects did not result in values different from those where com- plete abstinence was the case. The frequency distri- bution for 24-h protein excretion (Figure 5) suggests that most samples have a primary gaussian distribu- tion but that this overlaps with the distribution for a subgroup (17). We believe that this subgroup reflects active, vigorous young men with very mild, benign physiological proteinuria. Three young men were not included in the illustrated distributions because their daily total urinary protein excretion ranged from 257 to 387 mg and samples gave a trace reac- tion with the sulfosalicylic acid screening test. All values obtained, however, were included in the nonparametric normal range determination (18). Be- cause we know of no clinical conditions in which pro- tein excretion is subnormal, we determined the 95%
“Biuret:DTC method” is our abbreviation for the proposed method.
Table 3. Recovery of HSA Added to Three Clinical Urine Samples
HSA Total pro- HSA re. added tein detnd. coveredPresent in
sample mg/dl
Recovery %mg
A. 47.5 15.8 62.7 15.2 96 47.5 31.6 78.6 31.1 98 47.5 55.3 103.5 56.0 101
B. 45.6 15.8 61.2 15.6 99 45.6 31.6 78.4 32.8 104
45.6 55.3 101.0 55.4 100 C. 31.6 7.9 39.4 7.8 99
31.6 15.8 48.2 16.6 105 31.6 31.6 64.5 32.9 104
confidence limits for a one-tailed test. The normal range was 82 to 207 mg of total urinary protein ex- creted per 24 h.
Total protein concentrations in 40 clinical urine samples were determined in two groups of 20 on each of two days by the proposed biuret:DTC method; by the TCA:biuret method;8 by the gel filtration:biuret method;9 by micro-Kjeldahl analysis according to Ma and Zuazaga (20) of the initial gel effluent; and by the semi-quantitative sulfosalicylic acid test ac- cording to White et al. (21). Individual results are compared in Table 4. Results of Kjeldahl analyses were considerably higher in every case. Figures 6 and 7 are scatter diagrams around regression lines com- paring methods. Comparative statistics are summa- rized in Table 5. Contribution of native copper (cop- per bound to protein at loci other than peptide bonds) to the analyses for 24 clinical samples was 1
± 1%; results for 19 samples were 1% or less, and
8”TCA:biuret method” is our abbreviation for the method of Foster et a!. (19).
“Gel filtration:biuret method” is our abbreviation for the method of Doetsch (13).
55
in
102
I, a
Table 4. Comparison of Results for Total Urinary Protein Determinations in 40 Clinical SampIesz
Method
17 8 20 12 21 10 24 35
25 24
26 3
SSA6 (21)
neg neg neg neg neg neg trace trace trace trace neg trace trace trace trace trace trace 1+ 1+
1+ 1+
1+ 2+
17
Fig. 6. Scatter diagram around regression line comparing resultsby the biuret:DTC and TCA:biuret (19) methods
- in in
IDIOtPWtIi IWil
Fig. 7. Scatter diagram around regression line comparing results by the biuret:DTC and gel filtration:biuret (13) methods
only one result was greater than 3% (5% for one sam- ple). Total protein concentration of the “Abnormal Urine Chemistry Control” was determined to be 67 mg/dl, whereas manufacturer’s stated value was 70 ± 20 mg/dl, as measured by a turbidimetric method.
Total protein concentration of “Human CSF Con- trol,” Lot No. 2917-670H1 (Lederle Diagnostics, Pearl River, N. Y. 10965), was determined to be 80 mg/dl; the manufacturer’s stated value was 89 ± 11 mg/dl (turbidimetric).
Interferences
Removal of urinary pigments from seven clinical samples was 99 ± 1% (SD) complete. Recovery of HSA from two clinical samples demonstrating gross hemoglobinuria was 103% and 104%. Recovery of HSA from a clinical sample demonstrating gross bili- uria was 98%. Two urine samples and three different dilutions of another urine sample were assayed for total protein by the proposed biuret:DTC method. Then 5 ml of radiographic contrast medium (“Hypaque Meglumine 60% (w/v)”; Winthrop
35 40 46
76 77
88 100 108 134 181 190 191 200 238 242 255
289 289 291 298 310 313 425
638
22
84 578
O Routine hospital patient specimens, randomly selected. b Sutfosaticylic acid test.
CLINICAL CHEMISTRY, Vol.19,No.10,1973 1175
in in in
Fig. 5. Frequency data for total urinary protein excretion for 85 healthy adults
a
Table 5. Summary of Comparati X±SD Y±SD
ye b
m ,Reference method n mg/dl
TCA:biuret 40 120 ± 130 140 ± 137 33 73 -20 0.89 0.85 Gel filtration:biuret 40 150 ± 146 140 ± 137 1 29 +10 0.92 0.98
U Abbreviations for statistical parameters: n = no. samples b = y-intercept of regression line X = mean of reference method Sy = standard error of estimate Y = mean of test method m = slope of regression line
SD = standard deviation - -
Bias = mean of reference method (X) minus mean of test method (Y). r = correlation coefficient
Laboratories, New York, N. Y. 10016) was added to a 95 ml aliquot of urine of previously established pro-
tein concentration, the reaction mixture was allowed to stand at room temperature for about 1 h, and each sample was reassayed in triplicate. Total protein con- centrations upon re-assay fluctuated widely and un- predictably with both intra- and inter-samples, indi- cating gross interference.
Amount of Copper Bound to Protein
The protein-to-copper ratio was determined with CuSO4.5H20 standard. One milligram of copper is bound to 10.3 mg of HSA. One milligram of copper is bound to 10.1 mg of HSP. It was calculated that 1 mg of copper is bound to 9.9 mg of human serum gamma globulin. These factors are in accord with Nielsen (10). When human serum gamma globulin is measured against HSA standard by the biuret:DTC method, the maximum inherent error expected is 4%.
Using the value 611 amino acids per HSA mole- cule (22) and 69300 molecular weight, we calculated a ratio of six peptide nitrogen atoms per copper atom. This is in accord with Strickland et al. (23), but does not contradict the quaternary structure proposed for the protein-biuret complex (24). It is reasonable that the conformational change of HSA from its native elliptical shape to a random coil oc- curring in a highly alkaline medium only exposes two-thirds of the peptide bonds for reaction with cu- pric ion while the remaining ones are involved with maintaining the random-coil structure.
Discussion Sensitivityofthe Method
We determined comparable absorptivities (a), using a solution of HSA and a Beckman Model DU spectrophotometer.
Wave- Met hod length, nm
Biuret 550 4
Spectrophotometric 280 6
Lowry 660 174
Biuret:DTC 440 170
These data show that the proposed biuret:DTC method gives results that are comparable in sensitiv- ity to those obtained by the Lowry method.
Gel Filtrationand Linearity
Gel-filtration behavior of other protein species was identical to that of HSA, as determined by recovery
studies and by concentration vs. absorbance plots of dilutions of HSA, HSP, and four clinical urine sam- ples. The lag in the standard curve at low protein concentrations is an undesirable feature, but gel fil- tration appears to elicit a more diffuse elution band for protein solutions of very low concentrations- probably because of greater relative interactions of proteins with the solvent system and the dextran gel matrix-and lower recovery results. It is possible
that the constantly lower recovery of protein from the second gel filtration compared to the initial gel filtration is caused by increased nonspecific adsorp- tion of the highly negatively charged biuret complex to the gel matrix, similar to such adsorption of aro- matic compounds. It should be mentioned, however, that the lowest total protein concentration found in analyzing over 200 urine specimens was 3 mg/dl. Only six samples contained less than 5 mg/dl.
Although desalting methodology and the hygro- scopic nature of the gel make it possible to perform the gel filtration without constant attention, one should be aware of general principles of gel-filtration chromatography and not deviate from recommenda- tions provided by the manufacturer (14). Other dex- tran gels such a Sephadex G-25-80 may be used both for initial gel filtration and for second gel filtration to remove excess copper. All proteins and peptides having a molecular weight greater than 5000 can then be determined. Relative sample and eluent vol- umes for chromatography differ slightly and cost per test is higher. We have found that “Biogels” P-6 or P-b (200-400 mesh) (BioRad Laboratories, Rich- mond, Calif. 94804) may be used for the initial de- salting step but not for the copper removal step (there is no specific binding of the copper such as oc-
curs with the dextran gels). One should carefully in- vestigate proposed substitution of other reagents for gel filtration, because subtle differences in chromato- graphic behavior exist.
Method Comparison
Method comparison is difficult because we feel there is no acceptable reference method currently available to which the results of the new method can
CLINICALCHEMISTRY, Vol. 19, No. 10, 1973 1177
be validly compared. Some information, however, can be ascertained from the comparative data. The TCA:biuret method of Foster et al. (19) is widely
used. It should be realized that in addition to the presence of proteins in urine that do not precipitate in TCA (25-28), efficiency of protein precipitation decreases with decreasing protein concentrations and increasing heterogeneity of the biological fluid, ac- cording to the basic principles of precipitation analy- sis (29, 30). This accounts for falsely low results for urines with low protein concentrations. Low urinary protein concentrations are encountered when renal- transplant patients are monitored as an index of im- munological rejection (31). Falsely high results ob- tained for urines with high protein concentrations are explained by co-precipitation of chromogenic substances (29, 30, 32). The gel filtration:biuret method (13) suffers from a mild positive bias be- cause chromogenic substances can be adsorbed to protein and are thereby excluded from the gel and are able to contribute to the analysis. This positive bias is similar to the co-precipitation bias of the TCA:biuret method. Kjeldahl analysis gives valid re- sults only for 100% pure protein samples. No at- tempt to correct for nonprotein nitrogen was made in this study, resulting in a large positive bias.
General agreement of results determined by the different biuret methods is demonstrated (Figures 6 and 7), but we believe the most valid comparison among methods is between the proposed biuret:DTC method and the semi-quantitative sulfosalicylic acid screening test. The proposed method agreed more consistently with the sulfosalicylic screening test than did the other methods (Table 4).
For a discussion of statistical investigation of er- rors encountered in method-comparison studies, the reader is referred to a recent article by Westgard and Hunt (33). Comparative statistics and scatter di- agrams show that there was less variability for the methods in which gel filtration is used than for the method in which protein precipitation is used. The large constant difference between the results of the proposed method and those of the TCA:biuret meth- od (33 mg/dl) was expected, owing to inherent dif- ferences in methodologies. There was little constant difference (1 mg/dl) between the methods in which gel filtration is used. Proportional difference was greater with the TCA:biuret method (11%) than with the gel filtration:biuret method (8%). Inspection of the bias statistics shows that protein determinations by the proposed method are generally higher than by the TCA:biuret method, but generally lower than by the gel filtration:biuret method. Analytical biases of methods in which protein is precipitated and the problem of binding of interferences by protein have been discussed.
In principle, only random error cannot be elimi- nated. Systematic errors, however, can be reduced by improved methodology-a specificity problem is indicated by a large constant difference. We believe that, of methods currently available, our method is
the most specific for the peptide backbone chain of proteins.
Interferences
Interference from urinary pigments is virtually eliminated by the two gel filtration steps and by the high absorptivity of copper diethyldithiocarbamate complex (a = 15000). In the analysis of specimens containing hemoglobin, the hemoglobin was present in the initial gel effluent, but after treatment with biuret reagent, alkaline hematin was removed by the second gel filtration. Bilirubin not bound to protein was removed from specimens containing bile by the initial gel filtration. After treatment with biuret re- agent, the remaining bilirubin was removed by the second gel filtration. The radiographic contrast me- dium that we checked interferes with the biuret reaction (2) and also unpredictably affects results of the proposed modified biuret method. Freedom from interferences generally encountered in the clinical chemistry laboratory, however, is expected because substances can interfere only if they bind to protein and simultaneously chelate copper. This is expected to be the case with “Meglumine” (meglumine diatri- zoate).
Sodium diethyldithiocarbamate reagent is not completely specific for copper. Cations of iron, co- balt, cadmium, nickel, and bismuth are potential sources of interference (34). Masking by ethylenedi-
amine tetraacetate or citrate is not necessary be- cause the two gel filtration steps completely elimi- nate inorganic substances not bound to protein. Therefore only interfering cations bound to protein can result in analytical errors. Less than one atom of copper per molecule is usually attached to human serum albumin (22). Amounts of other divalent Ca- tions found are analytically insignificant. Calcula- tions from copper-binding data show that 102 atoms of copper are bound to one molecule of HSA in the course of the biuret reaction. Ceruloplasmin is rarely found in urine, owing to its high molecular weight and inability to pass the glomerular membrane. The contribution possible by transferrin is also negligible because of its low relative concentration and because it binds only two atoms of ferric iron per molecule. Interference by native copper and iron usually found in urine is therefore negligible.
One should be aware that falsely elevated results can occur in rare cases of physiological disturbance of copper metabolism, for example, in Wilson’s dis- ease. Moderate increase in urinary copper excretion also can occur in cirrhosis of the liver, particularly of the biliary type. Under standard dietary conditions, copper excretion fluctuates only slightly daily uri- nary copper excretion rarely exceeding 0.1 mg per day (35).
Amount of Copper Bound to Protein
It is important that the copper concentration of
the biuret reagent not be excessively high because secondary binding occurs (24, 36). Sodium hydroxide
1178 CLINICAL CHEMISTRY, Vol.19,No. 10,1973
concentration also must be carefully regulated. Base concentration should be at least 0.1 mol/liter, so that conformational changes of proteins from native states to random coils occur rapidly (3?).
In summary, the results of total protein assay by the proposed biuret:DTC method are not expected to be identical to those obtained by other methods, be- cause only the peptide chain backbone contributes to the analysis. Nonprotein substances found in the heterogeneous protein species contribute to dry weight analysis thereby affecting Kjeldahl factors and biuret coefficients. A set of copper-binding fac- tors for the different protein species, compared to dry weight analysis of pure samples, needs to be es- tablished.
The immediate value of the present method is sen- sitivity without sacrifice of specificity for quantita- tive analysis of total protein in biological fluids such as serum, cerebrospinal fluid, and urine, and for monitoring enzyme-purification steps.
This research was supported in parts by state-appropriated funds to the College of Graduate Studies and by the Trust Fund of the Department of Clinical Pathology of the Medical University of South Carolina, and by NIH GRS Grant No. 5S01 RR5420.
References 1. Martinek, R. G., Review of methods for determining proteins in biologic fluids. J. Amer. Med. Technol. 32, 177 (1970).
2. Caraway, W. T., and Kammeyer, C. W., Chemical interfer- ence by drugs and other substances with clinical laboratory test procedures. Clin. Chim. Acta 41, 395 (1972).
3. Elking, M. P., and Kabat, H. F., Drug induced modifications of laboratory test values. Amer. J. Hosp. Pharm. 25, 485 (1968). 4. Parvin, R., Pande, S. W., and Venkitasubramanian, T. A., On
the colorimetric biuret method of protein determination. Anal. Biochem. 12, 219(1965).
5. de Ia Huerga, J., Smetters, G. W., and Sherrick, J. C., Colon- metric determination of serum proteins: The biuret reaction. In Serum Proteins and the Dysproteinemias, F. W. Sunderman and F. W. Sunderman, Jr.,Eds. Lippincott, Philadelphia, Pa., 1964, pp 52-62.
6. Lowry, 0. H., Rosebrough, N. J.,Farr, A. L., and Randall, R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265(1951).
7. Zondag, H. A., and van Boetzlaer, G. L., Determination of protein in cerebrospinal fluid-sources of error in the Lowry method. Clin. Chim. Acta 5, 155(1960).
8. Pashley, D. H., Schuster, G. S., Palmer, P., and Sharaway, M. M., Some drugs and other substances that interfere with protein determination. Clin. Chem. 19, 263 (1973).
9. Peters, T., Jr., Proposals for standardization of total protein assays. Gun. Chem. 14, 1147 (1968). 10. Nielsen, H., Quantitative micro-determination of proteins and peptides. Acta Chem. Scand. 12, 38(1958).
11. Westley, J., and Lambeth, J., Protein determination on the basis of copper binding capacity. Biochim. Biophys. Acta 40, 364 (1960). 12. Klungsoyr, L., Quantitative estimation of protein-separation of alkaline protein-copper complex from excess copper on Sepha- dex G-25. Anal. Biochem. 27, 91(1969).
13. Doetsch, K., Determination of total urinary protein by com- bined gel filtration and automated biuret reaction. Clin. Chem. 18, 296(1972).
14. Sephadex-Gel Filtration in Theory and Practice. Pharmacia Fine Chemicals, Piscataway, N. J. 08854.
15. Weichselbaum, T. E., An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Amer. J. Clin. Pathol. 16 (Tech. Suppl.), 40(1946).
16. Callan, T., and Henderson, J. A., A new reagent for the color- imetnic determination of minute amounts of copper. Analyst (London) 54,650(1929).
17. Mainland, D., Remarks on clinical norms. Clin. Chem. 17, 267 (1971).
18. Reed, A. H., Henry, R. J., and Mason, W. B., Influence of statistical method used on the resulting estimate of normal range. Clin. Chem. 17, 275 (1971).
19. Foster, P. W., Rick, J. J., and Wolfson, W. Q., Studies in serum proteins VI: The extension of the standard biuret method to the estimation of total protein in urine. J. Lab. Clin. Med. 39, 618 (1952).
20. Ma, T. S., and Zuazaga, G., Micro-Kjeldahl determination of nitrogen. md. Eng. Chem. 14, 280(1942).
21. White, W. L., Erickson, M. M., and Stevens, S. C., Chemis- try for Medical Technologists. Mosby, St. LoUis, Mo., 1970, p 372.
22. Putnam, F. W., Structure and function of the plasma pro- teins. In The Proteins, 2nd ed., 3, H. Neurath, Ed. Academic Press, New York, N. Y., 1965, pp 154-267.
23. Strickland, R. D., Freeman, M. L., and Gurule, F. T., Copper binding by proteins in alkaline solution. Anal. Chem. 33, 545 (1961). 24. Mehl, J. W., Pacovska E., and Winzler, R. J., The amount of copper bound by protein in the biuret reaction. J. Biol. Chem. 177, 13 (1949).
25. Beckman, W. W., Hiller, A., Shedlovski, T., and Archibald, R. M., The occurrence in urine of a protein soluble in tnichloro- acetic acid. J. Biol. Chein. 148, 247 (1943).
26. Anderson, A. J., and Maclagan, N. F., The isolation and esti- mation of urinary mucoproteins. Biochem. J. 59, 638 (1955).
27. Popenoe, E. A., Characterization of a glycoprotein in the urine of patients with proteinuria. J. Biol. Chem. 217,61 (1955).
28. Saifer, A., and Gerstenfeld, S., Photometric determination of urinary protein. Clin. Chem. 10, 321 (1964). 29. Herman, J. A., and Suttle, J. F., Precipitation and crystalli- zation. In Treatise on Analytical Chemistry. part I, 3, I. M. Kol- thoff and P. J. Elving, Eds. Interscience, New York, N. Y., 1961, pp 1367-1406.
30. Salutsky, M. L., Precipitates, their formation, properties, and purity. In Treatise on Analytical Chemistry, part I, 1, I. M. Kol- thoff and P. J. Elving, Eds. Interscience,New York, N. Y., 1959, pp 733-766.
31. Harlan, W. R., Jr., Holden, K. R., Williams, G. M., and Hume, D. M., Proteinuria and nephrotic syndrome associated with chronic rejection of kidney transplants. New EngI. J. Med. 277, 769(1967).
32. Cadeau, B. J., and Malkin, A., Elimination of chromogenic substances from urine before protein determination by the biuret method. Clin. Chem. 17, 638(1971).
33. Westgard, J. 0., and Hunt, M. R., Use and interpretation of
common statistical tests in method comparison studies. Clin. Chem. 19,49(1973).
34. Pernin, D. D., Organic Complexing Reagents. Interscience,
New York, N. Y., 1966.
35. Beam, A. G., Wilson’s disease. In The Metabolic Basis of In- herited Disease, 3rd ed., J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, Eds. McGraw-Hill, New York, N. Y., 1972, pp 1033-1050. 36. Foster, J. F., Plasma albumin. In The Plasma Proteins, 1, F. W. Putnam, Ed. Academic Press, New York,. N. Y., 1960, pp 179-239.
Lowry Sensitivity and Biuret Specificity
Karl Doetsch and Richard H. Gadsden
A highly sensitive method with specificity for the peptidechain backbone was developed fordetermi- nation of total urinary protein. Interfering substances are removed by gelfiltrationand cupricionsare sto- ichiometricallybound to the peptidebonds of protein by the biuretreaction.Ion-exchange characteristics of the gel are neutralized, allowing protein-copper complex to be separatedfrom excess cupricionsby a second gelfiltrationstep.Copper bound to peptide bonds is colorimetrically determined by use of sodi- um diethyldithiocarbamate. Nonprotein substances do not interfereunless they simultaneouslybind to proteinand chelatecopper.As littleas 1 mg of total protein per deciliter can be determined in heteroge- neous biological fluids such as urine.
Additional Keyphrases: normal range #{149}peptide-bound Cu determined with Na diethyldithiocarbamate #{149}inter- method comparison
The diverse physical and chemical properties of the various kinds of human protein have resulted in a large number of analytical methods (1). Each method is affected by external interferences and by inherent biases. Because numerous and often unde- fined substances are present in biologic fluids, ana-
lytical conditions are not always well controlled. Re- sults obtained depend on the method rather than the actual protein concentration. Albumin is a simple protein comprised essentially of only amino acids. Many other protein species contain various amounts of carbohydrates, lipids, and other constituents, and these nonpeptide constituents often contribute non- specifically and erroneously to protein analysis. Some reactions have been considered specific for protein because of the high concentration of protein and the minimum concentrations of interferences present in serum. For example, the biuret reaction is highly susceptible to interference by nonprotein sub- stances (2-5). Besides pigments, any substance con- taining nitrogen, oxygen, or sulfur that is able to
From the Departments of Biochemistry and Clinical Pathology, Medical University of South Carolina, Charleston, S. C. 29401.
Received July 10,1973;accepted Aug. 13, 1973.
complex cupric ion may cause falsely elevated re- sults. The Lowry method has been shown to be even more susceptible to interferences (6-8). Besides pig- ments, any substance able to reduce the Folin phe- nol reagent may cause falsely elevated results.
A suitable method combining both sensitivity and specificity has been lacking for the determination of low concentrations of protein in biologic fluids. Pe- ters (9) proposed that the method of choice for total protein assay in clinical laboratories should be a ver- sion of the biuret reaction, the measurement to be made relative to that for pure bovine serum albumin (or human serum albumin) that previously had been standardized by Kjeldahl analysis. A modification of the biurt method comparable to the Lowry method
in sensitivity was originally described by Nielsen (10) and modified by Westley and Lambeth (11).
Klungsoyr (12) further modified the earlier methods by using gel filtration to remove excess copper from the biuret reaction mixture. None of these three methods, however, was adapted to clinical laborato- ry use.
A semi-automated biuret method for determina- tion of total urinary protein, in which gel filtration is used to remove interfering substances, has been de- scribed by Doetsch (13). The limit of sensitivity is 50 mg/dl total protein in the sample. In the proposed method, removal of unbound copper by an addition- al gel filtration step and subsequent colorimetric assay for copper bound to peptide bonds extends the limit of sensitivity to 1 mg of total protein per decili- ter of sample. Nonprotein substances do not interfere because only the peptide chain backbone (-RCH- CO-NH-) contributes to the analysis. The new and highly sensitive modified biuret assay described here is a true determination of protein peptide back- bone as suggested by Nielsen (10) for a standard ref- erence method.
Materials and Methods
60 g 40 g 15 g
5g 1 liter
2 Columns are prepared by cutting off the tip and reaming the
opening with blunt-end surgical scissors. The enlarged opening assures a more rapid and uniform flow. Columns and funnels are washed with a test tube brush and tap water at the end of the day and can be re-used indefinitely.
Biuret reagent should not be added to the collection cups be- fore the column effluent has been collected completely, because protein sometimes coagulates as a result of the initial high rela- tive base concentration that exists as the first few drops are col- lected.
We have found that distilled water may be used as the eluent in the second gel filtration instead of the sodium hydroxide re- agent.
Manual colorimetry can be performed by adding 2.0 ml of DTC color reagent to the second column effluents and measuring the color developed after 10 minutes but within one hour.
CLINICAL CHEMISTRY, Vol. 19, No. 10,1973 1171
oratories, San Mateo, Calif. 94401): Plastic column
chromatography racks and trays, Cat. No. 0904. Polyethylene column elements, 11 mm in diameter and 50 mm high, Cat. No. 0956. Polyethylene col- umn funnels, Cat. No. 0901.
Single-channel “AutoAnalyzer” system (Techni- con Corp., Tarrytown, N. Y. 10591).
Reagents
Only analytical reagent-grade chemicals and dis- tilled water were used.
Sephadex G-50-80 (Pharmacia Fine Chemicals, Piscataway, N. J. 08854). The gel was hydrated ac- cording to manufacturer’s directions (14). A large- bore, 10-ml serological pipette was used to charge the columns.
Biuret reagent. A modification of Weichselbaum’s reagent (15) was prepared. Final sodium hydroxide concentration was 0.2 mol/liter. Additional sodium potassium tartrate was used to prevent turbidity in the reaction mixture.
NaKC4H4O6.4H20 NaOH CuSO4.5H2O KI Distilled water, dilute to
Sodium diet hyldithiocarbamate color reagent. Two grams of the crystalline salt (Mallinckrodt Chemical Works, Jersey City, N. J. 07303) was dis- solved in 1 liter of distilled water. This was a 20-fold excess over minimum concentrations required for lin- earity. The reagent is stable indefinitely at room temperature. Five-tenths milliliter of “Brij-35” sur- factant (Technicon) was added per liter for continu- ous-flow colorimetry.
Sodium hydroxide reagent, 0.1 mol/liter. Human serum albumin (Dade Division, American
Hospital Supply Corp., Miami, Fla. 33152). Purity and char’acter of Lot No. PRS-410 were verified by ultracentrifugal analysis, cellulose-acetate electro- phoresis, and spectrophotometric absorption analysis according to the criteria of Peters (9). Total protein value of 7.8 g/dl was assigned by micro-Kjeldahl ni- trogen analysis (nitrogen value multiplied by the factor 6.25). HSA1 was determined to be more than 99.5% pure and to contain less than 5% aggregates. Working standards in the range 2-80 mg/dl were made by dilution with physiological saline. These standards are stable for about a month stored at 25 #{176}Cin brown bottles if a few crystals of sodium azide are added.
A bnormal urine chemistry control (Lederle Diag- nostics, Pearl River, N. Y. 10965), Lot No. 2921- 695H1, was diluted two-fold with physiological saline and stored as were the working standards.
1 Nonstandard abbreviations used: HSA, human serum al- bumin (Dade Division, American Hospital Supply Corp., Miami, Fla. 33152); DTC, sodium diethyldithiocarbamate; HSP, human serum protein reference standard (4.0 g of albumin and 3.5 g of y-globulin per deciliter; lot No. 2C4V; Dow Diagnostics, India- napolis, hid. 46206); TCA, trichloroacetic acid.
Procedure
Preparation of columns:2 A set of two columns is used for each standard, sample, and control. For ini- tial gel filtration, charge the columns with Sephadex
G-50-80 until the top of the gel is approximately 2 mm from the top of the column. For the second gel filtration, charge the columns with Sephadex G-50- 80 until the top of the gel is about 4 mm from the top of the column. (The gel expands when base is added.) Insert polyethylene funnels into the col- umns. Add 15 ml of sodium hydroxide reagent to each column to be used for copper removal in the second gel filtration. After the sodium hydroxide re- agent has passed through, this column is ready for removal of excess copper, because the weak ion-ex- change properties of the gel have been neutralized, thus preventing loss of copper on the gel. All col- umns are prepared freshly each day, but may then be used throughout the working day.
When a diluted aliquot of biuret reagent (contain- ing no protein) was treated by the copper removal step, the absorbance reading of the effluent to which
DTC reagent had been added was zero, indicating that excess copper is completely removed and that there is no copper in the column bleed.
Gel filtration: Apply 1.0 ml of working standards, samples, and controls to individual columns. Allow the liquid to percolate into the gel until column flow ceases. Remove the last hanging drop. Insert a 5-mi polystyrene AutoAnalyzer cup into the rack beneath the column and then gently apply 2.0 ml of distilled water to the top of each column. Collect the effluent until column flow ceases, including the last hanging drop. Add 0.5 ml of biuret reagent to each cup,3 mix well (mixing is conveniently accomplished by gently injecting air bubbles with a disposable Pasteur pi- pette), and allow to stand at room temperature. After 30 mm, apply 1.0 ml of the biuret reaction mixture to a base-conditioned column and allow to percolate into the gel until column flow ceases. Re- move the last hanging drop. Place a 5-mi polystyrene cup beneath each column and then carefully apply 2.0 ml of sodium hydroxide reagent4 to the top of each column. Collect the effluent until column flow ceases, including the last hanging drop.
Colorimetry: The second gel effluent may be ana- lyzed by manual5 or automated colorimetry. Figure 1
EwufNTII.
Fig. 3a. Elution profile (Sephadex G-50-80), first gel fil- tration
1.0
0.8
0.6
0.4
30/H
SAMPLER
To Sample, Wash Receptacle
Waste - p .056 FROM F/C
Fig. 1. Diagram of manifold for determination of total uri- nary protein by the biuret:DTC method
Fig. 2. Strip-chart recording, illustrating linearity, carry- over, and steady state
shows the manifold diagram. The working standards, samples, and controls are analyzed for copper by using the sodium diethyldithiocarbamate color re- agent (16). The yellowish-brown color is measured at 440 nm. Ninety-seven percent of full color develop- ment occurs within 3 mm, but the color must be measured within 1 h because the copper diethyldi- thiocarbamate complex is unstable. Decreasing per- cent transmittances of working standards are plotted vs. HSA concentrations. Sample and control values are read directly from the chart.6
Results Evaluation of Continuous-Flow Colorimetry
Variation of continuous-flow colorimetry for sam- ples run successively was less than 1%. Precision for
samples run in mixed orders and interaction between successive samples were acceptable. Figure 2 is a typical strip chart recording illustrating the standard curve, sample interaction, and steady state. Note that sample interaction was reduced by using DTC color reagent in place of distilled water in the Sam- pler II wash receptacle.
Gel Filtration and Linearity
The eiution profile for initial gel filtration (Figure 3a) was determined in fractions from the column by
6 We have found that the series of working standards can be
prepared in advance by individually pooling the gel effluents from several columns. These may be stored in brown bottles at room temperature and used to calibrate each run. A control, however, is included with each group of samples.
Fig. 3b. Elution filtration
use of the biuret reaction and a “Chloride Meter 920” (Corning Glass Works, Sci. Instruments Div., Medfield, Mass. 02052). At least 92% of salts and
small molecules are removed from urine by the first gel filtration step. The protein elution profile for the second gel filtration (Figure 3b) was determined by using DTC color reagent with column fractions.
Recovery from gel filtration of protein from solu- tions of various concentrations of HSA, HSP, and clinical urine samples is constant for different pro- tein species tested (Table 1), but reflects the phe- nomenon of less-efficient gel filtration behavior of very dilute protein solutions as compared to more concentrated ones. Recovery of protein from the first gel filtration step was determined by applying 1.0 ml
Added Determined Recovery
Added Determined Added Determined Recovery Recovery
mg/mi % mg/mi %
0.085 87 0.185 95 0.366 94 0.539 93 0.590 0.560 95 0.755 97 0.740 0.710 95 0.947 97 1.91 98 1.85 1.74 94 2.56 98 2.47 2.38 97 3.86 99 3.70 3.65 99 7.74 99 7.40 7.28 98
0.016 66 0.037 0.024 68 0.032 65 0.074 0.049 66 0.064 66 0.150 0.103 68 0.130 67 0.220 0.151 69 0.255 65 0.370 0.261 70 0.414 71 0.590 0.446 76 0.563 72 0.740 0.555 75
1.32 1.31
2.75 2.72
0.045 0.030
0.300 0.020
99
99
67
67
O Results of triplicate determinations. with use of Sephadex G-50.80. Human serum albumin (Dade Division). Human serum protein (Dow Diagnostics): 4.0 g albumin and 3.5 g gamma globulin per deciliter.
10
Fig. 4a. Standard curve for HSA, absorbance vs. concen- tration
Table 1. Protein Recovery from Gel Filtration’
CLINICAL CHEMISTRY. Vol. 19, No.10,1973 1173
mg/mi
2.60
3.90
7.80
0.049
0.098 0.195 0.390 0.585 0.780
of protein solution to a gel column and eluting with 2.0 ml of distilled water. A second 1.0-ml aliquot was
diluted to 2.0 ml with distilled water. Biuret reagent was added to both sets and, after 30 mm, absorban- ces were measured at 550 nm. Recovery of protein from the second gel filtration was determined by isolating the biuret protein-copper complex and sub-
sequently applying 1.0 ml of the ensuing protein- copper solution to a base-treated gel column. The el- uent used was 2.0 ml of sodium hydroxide reagent. A second 1.0-ml aliquot was diluted to 2.0 ml with so- dium hydroxide reagent. DTC color reagent was added to both sets and, after 10 mm, absorbances were measured at 440 nm.
A representative standard curve for HSA (absorb- ance) is illustrated in Figure 4a. The lag at low con- centrations results from the aforementioned gel-fil- tration behavior. The relationship between absorb- ance and protein concentration is essentially linear from 20 to 80 mg/dl. The lag problem in standard- ization is circumvented by the use of an AutoAnalyz- er chart reader whereby percent transmittance is plotted vs. HSA concentration (Figure 4b). By this technique, a straight line is obtained from 2 mg/dl to 20 mg/dl, the portion of the curve where sensitivi- ty is of greatest importance.
Precision
The largest coefficient of variation for within-run precision was 5%, and precision increased with pro- tein concentration (Table 2). Mean ± SD for three dilutions of the “Abnormal Urine Chemistry Con- trol” assayed once daily over 20 days was 16 ± 1, 33
± 2, and 67 ± 3 mg/dl. Best results were obtained by using glass pipettes.
Accuracy
Known amounts of HSA were added to previously
analyzed clinical urine samples. The results of recov- ery studies are presented in Table 3. Starting con- centrations of three clinical urine samples and of HSA solution added were determined by taking the mean value of 10 analyses. Various amounts of HSA solution were added to 100-ml aliquots of each urine sample, and the resulting samples then were ana- lyzed in triplicate. Recovery of added HSA was 101 ± 3%.
‘C
a
Protein concen- tration mg/dl
Fig. 4b. Standard curve for HSA, percent transmittance vs. concentration
B. Clinical urine samples
Normal Values
0.212 ± 0.002 0.426 ± 0.005
0.642 ± 0.006 0.850 ± 0.002
0.068 ± 0.001 0.107 ± 0.003 0.147 ± 0.001 0.293 ± 0.009 0.684 ± 0.009 0.934 ± 0.19
#{176}Results of quintuplicate determinations.
Coefficient of variation
5
2
3
3
3
2
Urinary 24-h protein excretion for 88 healthy white adults was determined over an eight-week period by
the biuret:DTC method.7 The range of total creati- nine excretion was 1.12 to 2.52 g per day. Subjects were volunteers from the student body and staff at the Medical University of South Carolina. Eighty were men between 22 and 36 years of age; three subjects were older than 36 years but younger than
55 years. Five subjects were women 22 to 29 years old. The values for all the women fell below the mean value, but there was no grossly discernible bias
because of age or sex. The medical histories of the subjects were screened by personal interview, ques- tionnaires, and examination of student health rec- ords. Consumption of all medications and of alcohol was controlled by requesting abstinence for 48 h be- fore and during the collection period. Small amounts of common medications (antihistamines, aspirin, oral contraceptives) taken by four subjects and small amounts of alcohol consumed by eight subjects did not result in values different from those where com- plete abstinence was the case. The frequency distri- bution for 24-h protein excretion (Figure 5) suggests that most samples have a primary gaussian distribu- tion but that this overlaps with the distribution for a subgroup (17). We believe that this subgroup reflects active, vigorous young men with very mild, benign physiological proteinuria. Three young men were not included in the illustrated distributions because their daily total urinary protein excretion ranged from 257 to 387 mg and samples gave a trace reac- tion with the sulfosalicylic acid screening test. All values obtained, however, were included in the nonparametric normal range determination (18). Be- cause we know of no clinical conditions in which pro- tein excretion is subnormal, we determined the 95%
“Biuret:DTC method” is our abbreviation for the proposed method.
Table 3. Recovery of HSA Added to Three Clinical Urine Samples
HSA Total pro- HSA re. added tein detnd. coveredPresent in
sample mg/dl
Recovery %mg
A. 47.5 15.8 62.7 15.2 96 47.5 31.6 78.6 31.1 98 47.5 55.3 103.5 56.0 101
B. 45.6 15.8 61.2 15.6 99 45.6 31.6 78.4 32.8 104
45.6 55.3 101.0 55.4 100 C. 31.6 7.9 39.4 7.8 99
31.6 15.8 48.2 16.6 105 31.6 31.6 64.5 32.9 104
confidence limits for a one-tailed test. The normal range was 82 to 207 mg of total urinary protein ex- creted per 24 h.
Total protein concentrations in 40 clinical urine samples were determined in two groups of 20 on each of two days by the proposed biuret:DTC method; by the TCA:biuret method;8 by the gel filtration:biuret method;9 by micro-Kjeldahl analysis according to Ma and Zuazaga (20) of the initial gel effluent; and by the semi-quantitative sulfosalicylic acid test ac- cording to White et al. (21). Individual results are compared in Table 4. Results of Kjeldahl analyses were considerably higher in every case. Figures 6 and 7 are scatter diagrams around regression lines com- paring methods. Comparative statistics are summa- rized in Table 5. Contribution of native copper (cop- per bound to protein at loci other than peptide bonds) to the analyses for 24 clinical samples was 1
± 1%; results for 19 samples were 1% or less, and
8”TCA:biuret method” is our abbreviation for the method of Foster et a!. (19).
“Gel filtration:biuret method” is our abbreviation for the method of Doetsch (13).
55
in
102
I, a
Table 4. Comparison of Results for Total Urinary Protein Determinations in 40 Clinical SampIesz
Method
17 8 20 12 21 10 24 35
25 24
26 3
SSA6 (21)
neg neg neg neg neg neg trace trace trace trace neg trace trace trace trace trace trace 1+ 1+
1+ 1+
1+ 2+
17
Fig. 6. Scatter diagram around regression line comparing resultsby the biuret:DTC and TCA:biuret (19) methods
- in in
IDIOtPWtIi IWil
Fig. 7. Scatter diagram around regression line comparing results by the biuret:DTC and gel filtration:biuret (13) methods
only one result was greater than 3% (5% for one sam- ple). Total protein concentration of the “Abnormal Urine Chemistry Control” was determined to be 67 mg/dl, whereas manufacturer’s stated value was 70 ± 20 mg/dl, as measured by a turbidimetric method.
Total protein concentration of “Human CSF Con- trol,” Lot No. 2917-670H1 (Lederle Diagnostics, Pearl River, N. Y. 10965), was determined to be 80 mg/dl; the manufacturer’s stated value was 89 ± 11 mg/dl (turbidimetric).
Interferences
Removal of urinary pigments from seven clinical samples was 99 ± 1% (SD) complete. Recovery of HSA from two clinical samples demonstrating gross hemoglobinuria was 103% and 104%. Recovery of HSA from a clinical sample demonstrating gross bili- uria was 98%. Two urine samples and three different dilutions of another urine sample were assayed for total protein by the proposed biuret:DTC method. Then 5 ml of radiographic contrast medium (“Hypaque Meglumine 60% (w/v)”; Winthrop
35 40 46
76 77
88 100 108 134 181 190 191 200 238 242 255
289 289 291 298 310 313 425
638
22
84 578
O Routine hospital patient specimens, randomly selected. b Sutfosaticylic acid test.
CLINICAL CHEMISTRY, Vol.19,No.10,1973 1175
in in in
Fig. 5. Frequency data for total urinary protein excretion for 85 healthy adults
a
Table 5. Summary of Comparati X±SD Y±SD
ye b
m ,Reference method n mg/dl
TCA:biuret 40 120 ± 130 140 ± 137 33 73 -20 0.89 0.85 Gel filtration:biuret 40 150 ± 146 140 ± 137 1 29 +10 0.92 0.98
U Abbreviations for statistical parameters: n = no. samples b = y-intercept of regression line X = mean of reference method Sy = standard error of estimate Y = mean of test method m = slope of regression line
SD = standard deviation - -
Bias = mean of reference method (X) minus mean of test method (Y). r = correlation coefficient
Laboratories, New York, N. Y. 10016) was added to a 95 ml aliquot of urine of previously established pro-
tein concentration, the reaction mixture was allowed to stand at room temperature for about 1 h, and each sample was reassayed in triplicate. Total protein con- centrations upon re-assay fluctuated widely and un- predictably with both intra- and inter-samples, indi- cating gross interference.
Amount of Copper Bound to Protein
The protein-to-copper ratio was determined with CuSO4.5H20 standard. One milligram of copper is bound to 10.3 mg of HSA. One milligram of copper is bound to 10.1 mg of HSP. It was calculated that 1 mg of copper is bound to 9.9 mg of human serum gamma globulin. These factors are in accord with Nielsen (10). When human serum gamma globulin is measured against HSA standard by the biuret:DTC method, the maximum inherent error expected is 4%.
Using the value 611 amino acids per HSA mole- cule (22) and 69300 molecular weight, we calculated a ratio of six peptide nitrogen atoms per copper atom. This is in accord with Strickland et al. (23), but does not contradict the quaternary structure proposed for the protein-biuret complex (24). It is reasonable that the conformational change of HSA from its native elliptical shape to a random coil oc- curring in a highly alkaline medium only exposes two-thirds of the peptide bonds for reaction with cu- pric ion while the remaining ones are involved with maintaining the random-coil structure.
Discussion Sensitivityofthe Method
We determined comparable absorptivities (a), using a solution of HSA and a Beckman Model DU spectrophotometer.
Wave- Met hod length, nm
Biuret 550 4
Spectrophotometric 280 6
Lowry 660 174
Biuret:DTC 440 170
These data show that the proposed biuret:DTC method gives results that are comparable in sensitiv- ity to those obtained by the Lowry method.
Gel Filtrationand Linearity
Gel-filtration behavior of other protein species was identical to that of HSA, as determined by recovery
studies and by concentration vs. absorbance plots of dilutions of HSA, HSP, and four clinical urine sam- ples. The lag in the standard curve at low protein concentrations is an undesirable feature, but gel fil- tration appears to elicit a more diffuse elution band for protein solutions of very low concentrations- probably because of greater relative interactions of proteins with the solvent system and the dextran gel matrix-and lower recovery results. It is possible
that the constantly lower recovery of protein from the second gel filtration compared to the initial gel filtration is caused by increased nonspecific adsorp- tion of the highly negatively charged biuret complex to the gel matrix, similar to such adsorption of aro- matic compounds. It should be mentioned, however, that the lowest total protein concentration found in analyzing over 200 urine specimens was 3 mg/dl. Only six samples contained less than 5 mg/dl.
Although desalting methodology and the hygro- scopic nature of the gel make it possible to perform the gel filtration without constant attention, one should be aware of general principles of gel-filtration chromatography and not deviate from recommenda- tions provided by the manufacturer (14). Other dex- tran gels such a Sephadex G-25-80 may be used both for initial gel filtration and for second gel filtration to remove excess copper. All proteins and peptides having a molecular weight greater than 5000 can then be determined. Relative sample and eluent vol- umes for chromatography differ slightly and cost per test is higher. We have found that “Biogels” P-6 or P-b (200-400 mesh) (BioRad Laboratories, Rich- mond, Calif. 94804) may be used for the initial de- salting step but not for the copper removal step (there is no specific binding of the copper such as oc-
curs with the dextran gels). One should carefully in- vestigate proposed substitution of other reagents for gel filtration, because subtle differences in chromato- graphic behavior exist.
Method Comparison
Method comparison is difficult because we feel there is no acceptable reference method currently available to which the results of the new method can
CLINICALCHEMISTRY, Vol. 19, No. 10, 1973 1177
be validly compared. Some information, however, can be ascertained from the comparative data. The TCA:biuret method of Foster et al. (19) is widely
used. It should be realized that in addition to the presence of proteins in urine that do not precipitate in TCA (25-28), efficiency of protein precipitation decreases with decreasing protein concentrations and increasing heterogeneity of the biological fluid, ac- cording to the basic principles of precipitation analy- sis (29, 30). This accounts for falsely low results for urines with low protein concentrations. Low urinary protein concentrations are encountered when renal- transplant patients are monitored as an index of im- munological rejection (31). Falsely high results ob- tained for urines with high protein concentrations are explained by co-precipitation of chromogenic substances (29, 30, 32). The gel filtration:biuret method (13) suffers from a mild positive bias be- cause chromogenic substances can be adsorbed to protein and are thereby excluded from the gel and are able to contribute to the analysis. This positive bias is similar to the co-precipitation bias of the TCA:biuret method. Kjeldahl analysis gives valid re- sults only for 100% pure protein samples. No at- tempt to correct for nonprotein nitrogen was made in this study, resulting in a large positive bias.
General agreement of results determined by the different biuret methods is demonstrated (Figures 6 and 7), but we believe the most valid comparison among methods is between the proposed biuret:DTC method and the semi-quantitative sulfosalicylic acid screening test. The proposed method agreed more consistently with the sulfosalicylic screening test than did the other methods (Table 4).
For a discussion of statistical investigation of er- rors encountered in method-comparison studies, the reader is referred to a recent article by Westgard and Hunt (33). Comparative statistics and scatter di- agrams show that there was less variability for the methods in which gel filtration is used than for the method in which protein precipitation is used. The large constant difference between the results of the proposed method and those of the TCA:biuret meth- od (33 mg/dl) was expected, owing to inherent dif- ferences in methodologies. There was little constant difference (1 mg/dl) between the methods in which gel filtration is used. Proportional difference was greater with the TCA:biuret method (11%) than with the gel filtration:biuret method (8%). Inspection of the bias statistics shows that protein determinations by the proposed method are generally higher than by the TCA:biuret method, but generally lower than by the gel filtration:biuret method. Analytical biases of methods in which protein is precipitated and the problem of binding of interferences by protein have been discussed.
In principle, only random error cannot be elimi- nated. Systematic errors, however, can be reduced by improved methodology-a specificity problem is indicated by a large constant difference. We believe that, of methods currently available, our method is
the most specific for the peptide backbone chain of proteins.
Interferences
Interference from urinary pigments is virtually eliminated by the two gel filtration steps and by the high absorptivity of copper diethyldithiocarbamate complex (a = 15000). In the analysis of specimens containing hemoglobin, the hemoglobin was present in the initial gel effluent, but after treatment with biuret reagent, alkaline hematin was removed by the second gel filtration. Bilirubin not bound to protein was removed from specimens containing bile by the initial gel filtration. After treatment with biuret re- agent, the remaining bilirubin was removed by the second gel filtration. The radiographic contrast me- dium that we checked interferes with the biuret reaction (2) and also unpredictably affects results of the proposed modified biuret method. Freedom from interferences generally encountered in the clinical chemistry laboratory, however, is expected because substances can interfere only if they bind to protein and simultaneously chelate copper. This is expected to be the case with “Meglumine” (meglumine diatri- zoate).
Sodium diethyldithiocarbamate reagent is not completely specific for copper. Cations of iron, co- balt, cadmium, nickel, and bismuth are potential sources of interference (34). Masking by ethylenedi-
amine tetraacetate or citrate is not necessary be- cause the two gel filtration steps completely elimi- nate inorganic substances not bound to protein. Therefore only interfering cations bound to protein can result in analytical errors. Less than one atom of copper per molecule is usually attached to human serum albumin (22). Amounts of other divalent Ca- tions found are analytically insignificant. Calcula- tions from copper-binding data show that 102 atoms of copper are bound to one molecule of HSA in the course of the biuret reaction. Ceruloplasmin is rarely found in urine, owing to its high molecular weight and inability to pass the glomerular membrane. The contribution possible by transferrin is also negligible because of its low relative concentration and because it binds only two atoms of ferric iron per molecule. Interference by native copper and iron usually found in urine is therefore negligible.
One should be aware that falsely elevated results can occur in rare cases of physiological disturbance of copper metabolism, for example, in Wilson’s dis- ease. Moderate increase in urinary copper excretion also can occur in cirrhosis of the liver, particularly of the biliary type. Under standard dietary conditions, copper excretion fluctuates only slightly daily uri- nary copper excretion rarely exceeding 0.1 mg per day (35).
Amount of Copper Bound to Protein
It is important that the copper concentration of
the biuret reagent not be excessively high because secondary binding occurs (24, 36). Sodium hydroxide
1178 CLINICAL CHEMISTRY, Vol.19,No. 10,1973
concentration also must be carefully regulated. Base concentration should be at least 0.1 mol/liter, so that conformational changes of proteins from native states to random coils occur rapidly (3?).
In summary, the results of total protein assay by the proposed biuret:DTC method are not expected to be identical to those obtained by other methods, be- cause only the peptide chain backbone contributes to the analysis. Nonprotein substances found in the heterogeneous protein species contribute to dry weight analysis thereby affecting Kjeldahl factors and biuret coefficients. A set of copper-binding fac- tors for the different protein species, compared to dry weight analysis of pure samples, needs to be es- tablished.
The immediate value of the present method is sen- sitivity without sacrifice of specificity for quantita- tive analysis of total protein in biological fluids such as serum, cerebrospinal fluid, and urine, and for monitoring enzyme-purification steps.
This research was supported in parts by state-appropriated funds to the College of Graduate Studies and by the Trust Fund of the Department of Clinical Pathology of the Medical University of South Carolina, and by NIH GRS Grant No. 5S01 RR5420.
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