1
Optical method and device for determination of protein and albumin
concentrations in human urine
A.Sünter1, A.Frorip2, V.Korsakov2, R.Kurrruk3, A.Kuznetsov2, M.Rosenberg1
1
- Tartu University; 2 – AS Ldiamon, Tartu Science Park; 3- Tartu, Family Medicine
Providers Mõisavahe Centre
[email protected]
Abstract
More than 260 samples of urine taken from the patients suffering renal failure,
mellitus diabetes, hypertension and some other diseases have been analysed in
the certified laboratory (Cobas 6000 Roche technique), with urine analyser H-50
(urine test strips) (DIRUI Industrial Co.) and with optoelectronic set-up
specially designed for this study. Both albumin and protein concentrations have
been determined and the data analysed. In the case with our special instrument
protein concentration was determined by the measurements of optical absorption
at 285 nm in the fractionated urines containing protein.
The main attention have been paid to the screening subgroup of 16 patients
having normal levels of albumin but enhanced levels of protein with the mean
0.17 and maximum 0.39 g/L. A fair correlation between maxima in protein
concentration as well as in protein/creatinine ratio values and diseases (renal
failure etc.) in this group has been noted. Comparison of results obtained by the
proposed optical method of protein determination and by the use of urine test
strips gives the advantage to our method in the case of this “normal albumin
group”.
Introduction. There exists a need for cheap and express methods for control of
albumin and protein levels in urine of persons suffering some diseases (kidney
failure, hypertension, diabetes mellitus etc.). Convenient steady control for
normal people in home environment (self-performed screening) is also an
important topic. Urine test strips (or dipstics) are well known instruments
invented for this purpose but they cannot enable sufficient precision. Moreover,
they are envisaged mainly for selective detection of albumin and are not
sensitive to some other proteins, e.g., Bence-Jones proteins and give negative
false results in this case. At the same time, just proteinuria disease is closely
correlated with diabetes and this fact directly points to one of a numerous focus
group among population for screening and straightforward control.
2
Aim. The aim of the undertaken study was to demonstrate the possibility to
exploit the UV absorption of proteins (≈285 nm) in fractionated urine for
estimation of their concentration. The second goal of the project was the
comparison of these results with those obtained in parallel by use of urine test
strips. Thirdly, a preliminary analysis from the clinical point of view was also
undertaken. The given research is a continuation of our previous one [1].
Method and patients. We propose a simple UV absorption measurement
method in a sensor with 5 mm path length cuvette of fractionated urine for
determination of proteins concentration. For such a fractionation commercially
available and cheap PD-10 desalting columns (GE Healthcare) with the cut-off
M < 5 kDa can be used [2]. The urine fraction eluted as the first fraction
contains pool of proteins (albumin included) with masses M > 5 Da. Some large
peptides can also be present in this fraction.
Many trials have been done to find out the best buffer applicable for
fractionation, its volume, reproducibility of results and other details. We have
observed that both the acidic (~pH4) and basic (≥ pH8) buffers are pretty good
for this purpose with a slight preference to basic ones. It seems that with basic
buffers one and the same column PD -10 can be used for a larger number of
fractionations (up to 70).
The absorption measurement takes place at λ = 285±5 nm in the first absorption
band of proteins (Fig. 1). The intensity of this absorption is in the linear
proportion to the protein concentration in a wide range up to 10 g/L.
Absorption of an example
of urine protein fraction
4.5
4
3.5
OD, 5 mm
3
2.5
2
1.5
1
0.5
0
200
250
300
nm
350
400
3
Fig. 1. Absorption spectrum and optical density of a high protein concentration 5
mm thick layer of urine fraction
The sensor has a software which enables the operator to monitor the evolution of
fractionation procedure in an on-line mode. The time interval actual for
registration of the first protein peak is approximately 45 sec. For enhancement
of the measurement precision we have made use of the total area under the
rotein peak (Fig. 2).
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
0:00
0:01
0:02
0:03
0:04
0:05
0:06
0:07
0:08
0:09
0:10
0:11
0:12
0:13
0:14
0:15
0:16
0:17
0:18
0:19
0:20
0:21
0:22
0:23
0:24
0:25
0:26
0:27
0:28
0:29
0:30
Optical density, a. u.
Urine fractions optical density calculated from
sensor readings
Time, min
Fig. 2. Fractionation chronogram of albuminuria urine in a desalting column
PD-10. The 5 min elution peak belongs to proteins with M > 5 Da.
Approximately 150 different urine samples were assayed in the laboratory of the
Tartu University Hospital (TUHL) (Cobas 6000 Roche systems equipment is
being used there) in relation to the albumin and protein concentrations and, in
parallel, the most part of them was fractionated and the absorption at 285 nm
measured. At the last end the calibration in the scales “area under the protein
peak in a chronogram versus protein concentration in the whole urine” was
obtained (Fig. 3) and used for further work.
4
10
9
y = 0.9917x + 0.2216
R² = 0.9681
8
Protein elution peak area, a.u.
7
6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
Protein concentration in whole urine, g/L)
Fig. 3. Correlation between the absorbed light sum in the protein elution peak
and concentration of protein in whole urine established for the largest range of
concentrations up to 10 g/L
The concentration of albumin was calculated with use of the empirical relation
y = 1.15x + 0.153 (1),
5
where y is the concentration of protein and x – concentration of albumin (all in
g/L) which was established on the base of a large number (133) of the TUHL
assays with different whole urines (Fig. 4).
Fig. 4. Correlation between albumin and protein in whole urines in the wide
range of concentrations for 133 on spot samples.
We have used a commercial DIRUI urinalysis instrument H-50 for the parallel
work with the samples under investigation. In this photoelectric colorimetry
device the urinalysis strips of the type H13 were used for an automated reading
of coloured strips at six possible wavelengths 400, 520, 560, 610, 660 and 940
nm.
Patients formed two groups. One group consisted of the TÜ nephrology Prof.
Rosenberg’s patients. The second group constituted the population
representatives (41) with diabetes, hypertension and other diseases which are
being observed at the Family Medicine Providers Centre Mõisavahe (Tartu).
Altogether 261 samples of urine were collected and analysed. All urines were
taken on spot and no 10-hours or day&night collections were undertaken.
Results and Discussion. In Fig. 5 we demonstrate the dependence of the ratio
albumin/protein in per cents on albumin concentration in 133 samples of whole
urines taken in both donators’ groups. Albumin and protein concentrations were
detected in the wide range extending from the lowest level of detection (0.002
and 0.01 g/L accordingly) up to approximately 10 g/L. This range can be
tentatively divided into three sub-regions: albuminuria (300 mg/L – 10 g/L),
microalbuminuria (30-300 mg/L) and normal level albumin region (< 30 mg/L).
6
Alb/Pr ratio, %
The distribution of data in Fig. 4 can be fairly approximated by the logarithmic
plot y = 11.33ln(x) + 69.89, where x is the concentration of albumin in g/L and y
– ratio Alb/Prot in %.
100
90
80
70
60
50
40
30
20
10
0
y = 11.331ln(x) + 69.886
R² = 0.834
0
2
4
6
Albumin, g/L
8
10
Fig. 5. Albumin/protein ratio in dependence on the albumin concentration in
whole urines
The distribution has a prominent break at the albumin concentrations near 300
mg/L, i.e., in the region between albuminuria and microalbuminuria. In the
region of albuminuria the ratio Alb/Prot asymptoticaly approaches the value 1
showing the decreasing specific part of proteins other than albumin.
On the contrary, in the region <300 mg/L the dependence has a character of
steep decrease in the direction to lower albumins. This means the increasing part
and role of not-albumin proteins in microalbuminuria and, especially, in the
range of seemingly normal urines (<30mg/L).
Fig. 6 illustrates more concretely what we are speaking about in relation to
proteinuria.
7
Alb&Prot distribution in urines with normal albumin
0.45
0.03
0.4
Protein, g/L
0.3
0.02
0.25
0.015
0.2
0.15
0.01
0.1
Albumin, g/L
0.025
0.35
0.005
0.05
0
0
0
2
4
6
8
10
12
14
16
18
Patients' number
Protein
Albumin
Linear (Protein)
Linear (Albumin)
Fig. 6. Distribution of albumin and protein in whole urines taken from 16
persons suffering renal failure, diabetes or/and hypertension with numbers
growing in the direction of albumin concentration increase. Only persons with
normal albumin level were selected. Concentrations are determined with the
Cobas 6000 Roche systems.
In Fig. 6 is depicted the distribution of albumin and protein levels in whole
urines collected from the people suffering renal failure, diabetes and/or
hypertension. Present 16 persons with normal albumin concentrations
(≤30mg/L) have been sorted out of 41 people strong cohort observed at the
Mõisavahe family medicine providers centre and formatted as a group for the
special thorough consideration. It is worth to mention that such a correlation
between albumin and protein as in Fig. 6 is free from the dependences of solutes
concentrations on the urine specific density and other altering factors which can
influence the results essentially when we are dealing with urines taken only once
on spot [3].
One can see in Fig. 6 that two distributions differ remarkably. Due to the used
illustration mode the albumin concentration distributes practically linear
whereas the distribution of protein fluctuates strongly. Really, its distribution
has also a slightly increasing trend as albumin does but it has not much
significance in the given aspect.
On the contrary to the albuminuria region the concentration of protein is much
higher than that of albumin in the mean values as well as in amplitudes. For the
collection of Fig. 6 albuminmean is 0.0127±0.0083 mg/L and proteinmean is
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0.1719±0.1003 g/L, i. e., the difference is stronger than 13.5 times. The
amplitude difference can be even higher and is, e.g., for the patient 3 66.7.
For the same patients’ group as in Fig. 6 similar distributions for ratios
Albumin/Creatinine and Protein/Creatinine can be drawn (Fig. 7).
3.5
0.25
3
0.2
2.5
0.15
2
1.5
0.1
1
0.05
0.5
0
Prot/Creat, g/g
Album/Creat, mg/mmmol
Distribution of Album/Creat and Prot/Creat
0
0
5
10
15
20
Patients' number
Alb/Creat
Prot/Creat
Fig.7. Ratios albumin/creatinine and protein/creatinine in urines from the same
patients as in Fig.6. Note that the patients’ numbers in Figs. 5 and 6 coincide
with each other only partially (for details see Table 1).
Ratios albumin/creatinine and protein/creatinine are often recommended to be
used instead of albumin and protein concentrations to get more stable and
reproducible results (see, for instance, [3] and references therein). We see in Fig.
7 that the ratio protein/creatinine fluctuates strongly on the background of
smoothly changing albumin/creatinine curve similar to the behaviour of albumin
and protein concentrations in Fig. 6. Moreover, some patients’ positions in both
rows at x-axis coincide with each other: 1, 2, 3, 7, 8, 9 (see Table 1). The first
and second maxima in Figs. 6 and 7 belong to the same patients 3 and 7 and the
patient 12 induces the maxima in Fig. 6 as well as in Fig.7 under the position 15
in Fig. 7.
We have noticed in Table 1 also the diseases for four patients (predominantly it
is renal failure). One can see that the maxima in the protein and
protein/creatinine distributions in Fig. 6 and 7 belong to the sick people. It is
remarkable that the protein distribution is more precise in this relation than its
counterpart protein/creatinine which shows one false maximum for the patient
10 (Fig.7). One of the reasons for that can be the lowered accuracy in
determination of creatinine concentration in biological fluids [4, 5].
9
Table 1. Coincidence and differences in patients’ positions in Figs. 6 and 7. The
distribution in Fig.6 is taken as the basic. By red are marked the maxima
positions. Diseases (renal failure and other) are marked only for the cases other
than hypertension and diabetes
Pat.
num.
in Fig.6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Pat.
num.
in Fig.7
1
2
3
5
6
4
7
8
9
11
13
10
15
16
12
14
Disease
Other
Ren.fail
Ren.fail
Ren.fail(12)
Ren.fail(12)
One should stress once again that all persons in the group being scrutinised here
have the lowest or normal level of albumin in urine. Therefore we will
concentrate our further attention to protein.
The Cobas 6000 Roche systems mark the ratio “protein/creatinine” > 0.200
mg/mg or >22.6 mg/mmol as the reference value for indication of possible
proteinuria. In Fig. 7 and in Table 1 one can see that only the patient 15 has the
characteristics pointing to the sickness, the other persons seemingly satisfy the
healthy status norm.
Nevertheless, it is possible to make use of the mean (0.099) or median (0.0845
mg/mmol) values in this group and consider the numbers higher than these as
(sub)-pathological levels or as ones suitable for early warning of proteinuria. For
three sick persons (3, 7, 10; Fig.6) these values turn out to serve not only for
warning but indicate their real sickness.
The same logic holds also for the concentrations of values for protein only. The
mean and median values are in this case 0.17 and 0.14 g/L, accordingly, and all
10
sick people have the higher levels than the median value and only the patient 9
with 0.15 g/L is under the mean.
We can add to this observation the fact that in a much larger group of 57 patients
with albumin < 30 mg/L there were the values: the mean - 0.096 g/L and median
– 0.07 g/L.
It is useful to compare both protein values 0.14 and 0.17 g/L with the free
members in the empirical equations of type (1) (see Fig. 4) compiled for mutual
concentrations of albumin and protein. Equation (1) is valid for the large number
(133) of urines with very different concentrations of albumin and protein from
the lowest measured values up to ~10 g/L. In this case the free member is 0.153
g/L.
We have done the similar manipulation for the urines collection (54 samples)
with normal albumin (<30 mg/L) and get the equation y = 5.646x + 0.043 (2).
The equation 2 shows more strong interdependence of protein (y) and albumin
(x) and the free member has the lower value (0.043) than in the eq.1 (0.153 g/L).
This difference gives evidence that for the correct estimation of albumin or
protein by using the equations 1, 2 (or similar) one has to take into account the
region where the estimation does take place: albuminuria, microalbuminuria or
seemingly “normal albumin” situation. The “normal” case can be a difficult
challenge and needs, to our opinion, further elucidation.
The positive sign of the free member in the equations of type (1) means that
concentration of protein is always higher than that of albumin and cannot be
much lower than 0.04 g/L. This level can be estimated as the normal or
endogenous level of urine proteins. The protein levels higher than 0.04 and,
certainly, higher than 0.1 g/L could be considered as warning levels. Just for
such levels there is a need for sensitive and simple express determination
methods and tools.
As the main result of this pilot study we present Table 2 with comparison of
results achieved with three determination methods: in the certified laboratory of
the TUH, with urine test strips (H-50 Urine Analyser) and with AS Ldiamon
sensor designed for the present project. Only the screening representatives of
population (16) with low (normal) albumin are embraced with this comparison.
One can see that in the most cases the test strips method failed to indicate any
protein even in the cases of its rather high level, e.g., 0.39 g/L (patient 15). At
the same time our UV sensor gave promising result and indicated protein in the
most cases besides two low concentration cases (Pts. 2 and 14) and, surprisingly,
for Pt. 2 with 0.2 g/L. The last situation is now under additional investigation.
11
We are analysing also the possibilities to enhance the protein determination
precision.
Table 2. Comparison of results obtained for a screening focus group with low
albumin in the TUH certified laboratory (Cobas 6000 Roche systems), with
urine test strips and with the AS Ldiamon sensor
Patient
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Test
Ldiamon
Labor.
Labor. strip
sensor
albumin, protein, protein, protein,
g/L
0.002
0.003
0.003
0.0049
0.0056
0.0063
0.0099
0.0115
0.0149
0.0155
0.0156
0.0182
0.0186
0.0218
0.0254
0.0275
g/L
0.11
0.07
0.2
0.09
0.08
0.13
0.29
0.09
0.15
0.1
0.18
0.36
0.24
0.1
0.39
0.17
g/L
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.15
0
g/L
0.08
0
0
0.06
0.03
0.08
0.2
0.03
0.28
0.06
0.2
0.1
0.05
0
0.37
0.37
Disease
Hypertension
Other
Hypertension
Hypertension
Ren. failure
Hypertension
Ren. failure
Hypertension
Hypertension
Ren. failure
Hypertension
Hypertension
Hypertension
Diab+hyperten
Conclusion. It seems that the reference values for proteinuria adopted in some
certificated laboratories can turn out to be too high in the case of patients’
groups with normal albumin levels in urine and having at same time elevated
concentrations of protein which can correlate with pathologies (renal failure
etc.) The matter needs further investigation and clarification.
The use of urine test strips usually designed for the selective albumin assays can
be not sufficiently effective in the case of the low urine albumin (<30 mg/L) and
appearance of enhanced level of proteins (0.15-0.4 g/L). In this case the need for
express screening methods of population for direct evaluation of protein
concentration can be especially actual. Proposed here method of measurements
of the UV optical absorption (285 nm) in urines fractionated in desalting
columns could be used for this purpose after the trials in the larger patients’
groups.
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Acknowledgments. We thank Mrs. Mare Säde for very useful participation in
this work.
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Tartu, September 20-22, 2012, p.51;
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