OPTICAL PROPERTIES OF URINE, BLOOD
PLASMA AND PULMONARY CONDENSATE OF THE
PATIENTS WITH PULMONARY FORM OF
TUBERCULOSIS
O.G. Uchenko, S.G. Guminetsky, A.V. Motrich.
Chernivtsi National University named after Yuriy Fedkovych
It is determined that the patients with pulmonary form of tuberculosis with cavityforming within the interval of the spectrum   250 nm the high values of relative
optical density both in the spectra of urine absorption and in plasma are observed.
The possibility to use the absorptional spectral analysis for determining the absolute
values of urea and uric acid concentration in human urine is substantiated. It is
shown that the value of optical density D in the area   285 nm for urine and
  280 nm for plasma can be regarded as optical test for defining the activity of
the process and the way of cure efficiency control. The results of spectrophotometric
investigations of the pulmonary condensate samples of the sound people and of the
patients with pulmonary form of tuberculosis within the spectral interval
215    340 nm are presented. The sufficient difference between then is
determined both in the content of biologically active substances and free aminoacids
(tryptophan and phenylalanine) is determined.
Key words: tuberculosis, urine, blood plasma, pulmonary condensate, optical density, absorption
spectrum.
1. INTRODUCTION
Human urine and blood plasma – are very complicated biological media to be
analyzed. The reason of this lies in multicomponentness of organic substances and
unstability of their concentrations during the day (especially for urine), depending
on the individual, his/her general state, type of food, etc. The concentration range
of each of the principal components of urine and plasma of the sound person
(normal) was determined by biochemical methods.
It is known that the main contribution in spectral absorption of urine in the area of
  260 nm is made by urea and uric acid only, while for plasma the absorption
spectra of proteins (albumins and globulins) play the significant part [1,2]. In this
article the absorption spectra of blood and morning portion of urine of the grownup patients with distructive tuberculosis of lungs within the spectral interval
215    340 nm are investigated. The changes in the process of standard
etiopathogenetic cure during 1-5 months are analyzed with the purpose to use the
obtained results for determining active process and the cure efficiency control.
In practical medical investigations there appear more and more research works on
using the humor of the air that is breathed out by the human lungs (pulmonary
condensate) for diagnostics of the diseases of bronchial system , pre-asthma,
ischemic heart disease and other non-specific chronic diseases [3,5]. However,
there are no data on the analysis (using the optical techniques, in particular) of
pulmonary condensate of the patients with pulmonary form of tuberculosis in the
literature. The results of analysis of absorption spectra of the pulmonary
condensate of the sound people (as test ones) and the patients with tuberculosis on
the same spectral interval 215    340 nm are given below. The possibilities of
using these spectra for determining the content of biologically active substances
(BAS) as well as the content of the aromatic aminoacids are also considered.
2. THE OBJECTS AND THE TECHNIQUE OF INVESTIGATION
The objects of our investigation are urine, plasma and pulmonary condensate of
the patients with the destructive tuberculosis of lungs. The routine laboratory
analyses of the majority of the patients without the concurrent kidney pathology
show that the quantity of proteins in urine is insufficient and it has practically no
influence on its optical properties. This makes the urine analysis much more easy
both from optical and biochemical point of view. The samples of pulmonary
condensate for the investigation were prepared according to the typical technique:
4-5 ml of condensate of the sound people and 2-3 ml of the tuberculous patient
had been gathered for 15 minutes in the device for obtaining condensate.
The measuring of the absorption spectra of the morning samples of urine, plasma
and pulmonary condensate were performed on the spectrophotometer СФ-4А with
photometric sphere according to the technique [1,6] which enables to consider the
light scattering, as the media under study are colloidal solutions. Urine and plasma
were diluted with distilled water in the proportion 1:100. The solutions under
study were places in quartz pan with the thickness l = 1 cm. The coefficient of
light transmission  was measured, and the optical density was calculated by the
formula D   lg  . The experiments prove that the chosen dilution of urine and
plasma are optimal for providing the accuracy of measurements at   340 nm
[2]. According to [1,2] the main contribution to the value of D for urine in shortwave maximum is made by urea (the rest of components amount to ~1.5% of the
total density value). That’s why the spread in measured values for D for different
patients at   235 nm is explained by that very fact that their concentration of
urea is different. To estimate the real contribution of uric acid in the total optical
density of urine in the area   250 nm, it is convenient to standardize the spectra
for every case by the values of optical density in maximum, which is found at
  235 nm. So, for further analysis we use only standardized values of D. The
same procedure is used for the analysis of plasma absorption spectra.
3. THE ANALYSIS OF ABSORPTION SPECTRA OF URINE AND
PLASMA
In [1] the absorption spectra of albumin and globulin as the main organic
components determining the absorption spectrum of plasma are studied. It is
determined that in the area of   280 nm the value of D is defined, in general, by
absorption of all the globulin, while in the area of   235 nm – by the total
concentration of all proteins. To estimate the interrelation between albumin and
globulin in plasma, we standardize their absorption spectra by the value of D in
short-wave maximum. Figure 1 (for urine) and Figure 2 (for plasma) present the
absorption spectra of three patients with pulmonary form of tuberculosis before
treatment in comparison with test absorption spectra (Curve 1) for a healthy man.
It can be seen from Fig. 1 that optical density of urine samples in the spectral
interval   265  295 nm for all three patients is much higher in comparison with
the test one. As the main part of the optical density value in this spectral interval
belongs to absorption of uric acid, then in the patients urine there is either the
increase of uric acid concentration, or its portion in the total content of urea and
uric acid. It can be observed that this effect is manifested in a different way for
various patients before treatment: Curves 2,3,4 in Fig. 1 are also different. Similar
situation is observed in the plasma absorption spectra, but this is just a superficial
resemblance. Actually, the reason of the difference between the D spectra for
plasma (Fig. 2) has quite a different nature and is explained by the fact that the
tuberculosis patients have a sufficiently changed value of albumin / globulin
coefficient. The clinical data obtained by biochemical methods prove that this
coefficient is about 1.8-2.0 for healthy men, while it is sufficiently lower for
tuberculosis patients. This demonstrates the increase of the portion of globulin in
the total content of proteins in plasma and, thus, leads to increase of the D/Dmax
values in comparison with the test Curve 1. Besides, before treatment the changes
in albumin / globulin coefficient is different for every patient: Curves 2,3,4 in the
spectrum interval   265  295 nm are different too.
The absorption spectra of urine and plasma of the patients with pulmonary form
of tuberculosis in the process of treatment enable to perform the investigations
with certain intervals, monitoring the changes of optical density. For example,
Fig.3 (for urine) and Fig. 4 (for plasma) present the standardized absorption
spectra for two patients obtained before treatment (Curves 1) and after a month of
treatment (Curves 2), and for urine – after 5 months of therapy (Curves 3).
It can be seen that in all the cases in the interval of   265 nm the relative optical
density decreases. This can be explained by the changes in relation between uric
acid and urea in urine and by the changes in the content of albumin and globulin
in plasma. At the same time, comparing the Curves in Fig. 3 and Fig.4 with the
Curve 1 in Fig. 1 and 2 respectively for the test samples, one can see that though
the value of D/Dmax decreased after a month of treatment, it is still far from
normal value for urine and plasma of the healthy man. After five-month’s
treatment this value in the majority of cases for urine tends to the test sample.
Similar results are obtained for the rest of the patients (12 patients), the samples of
urine and blood of which were presented for investigation. To generalize the
results it is enough to compare the values of relative optical densities for one, the
most typical wave-length. Using the above-presented graphs we choose   285
nm for urine and   280 nm for plasma.
Table 1
The D/Dmax values for urine of the patients with the pulmonary tuberculosis for
  285 nm (test value is 0.1)
patients
Time of checkup
K.
Before cure
a month’s treatment
5-month’s treatment
B.
U.
D.
K.
D.
C.
K.
T.
B.
Ch. M.
0.48 0.44 0.38 0.43 0.46 0.44 0.42 0.38 0.52 0.46 0.33 0.33
0.42 0.39 0.34 0.38 0.44 0.26 0.28 0.36 0.36 0.26 0.3 0.39
0.43 0.26
0.27
0.3
The data (for 10 patients) presented in Tables 1, 2 completely confirm the
conclusions made on the basis of the above presented graphs with the exception of
patient M. (urine) and patient Ch. (plasma). Besides, the tables prove that the
decrease of the D/Dmax values after a month’s treatment is different for every
patient. This can be explained by the individual peculiarities of every patient and
proves the efficiency of the cure. Specifically, Table 1 presents the values of
D/Dmax for urine of four patients after 5-month’s treatment. It is shown that two
of them have the further decrease of this value while the rest present practically no
change.
Table 2
The D/Dmax values for plasma of the patients with the pulmonary tuberculosis for
  280 nm (test value is 0.26)
Patients
Time of checkup
K.
B.
U.
D.
K.
D.
C.
K.
C.
B. Ch.
Before cure
0.43 0.48 0.45 0.46 0.42 0.42 0.44 0.47 0.53 0.42 0.43
a month’s treatment
0.37 0.44 0.37 0.32 0.39 0.33 0.37 0.34 0.44 0.37 0.43
4. SPECTROPHOTOMETRIC METHOD OF DEFINING THE
CONCENTRATION OF UREA AND URIC ACID IN URINE
To make the analysis of urine we consider the two-component approximation:
urea – uric acid. Let’s use the spectral dependencies of the extinction values of
these components presented in [2], from which we can conclude that   230 nm
(for urea) and   290 nm (for uric acid) can be considered the analytical wavelengths. We make up two equations for these values of :
D1   1,c cc l   1,ck cck l
(1)
D 2   2,c cc l   2,ck cck l
Here:  1c and  2c – are the values of urea extinction for the first and the second
analytical wave-lengths;  1,ck and  2,ck – for uric acid respectively; c c and c ck –
the required concentrations of urea and uric acid; l – the thickness of the pan,
which equals 1 cm in this case.
Solving the system of equations (1) we obtain:
 1,ck D 2   2,ck D1
 1,ck  2,c l   2.ck  1,c l
 1,ck  1,c D 2   2,ck  1,c D1
D
 1  2
 1,ck l  1,ck  2,c l   1,c  1.ck  2,ck l
Cc 
(2)
C ck
(3)
Table 3
The values of  for urine of the patients with pulmonary tuberculosis
Time
checkup
Before cure
a month’s
treatment
5-month’s
treatment
patients
of
K.
B.
U.
D.
K.
D.
C.
K.
C.
B.
Ch.
M.
M.
C.
T.
1.77 1.48 1.23 1.35 1.73 1.46 1.35 1.46 2.43 1.58 1.07
2.62
1.75 3.56 1.9
1.41 1.25 1.03 1.11 1.62 0.75 0.76 0.84 1.1 0.62 0.95
1.9
0.77 1.07 1.08
1.31 0.69 1.02
0.75
1.01
1.02
Using these formulas it is possible to calculate the concentrations of urea and uric
acid in real biological medium – human urine. For this purpose we take the values
of D 1 and D 1 from the curves D=F(), and from the [9] – the values of the
extinction for corresponding wave-lengths.
However, from the point of view of treatment efficiency estimation, it is not the
absolute value of Cc and Cck, that is important, but the relation of Cck to Cc, which
will physically characterize the changes in interrelation between the urea and uric
acid concentrations in the process of treatment. Let’s introduce the denotation
C ck / C c   .The calculated values of  for urine of the above mentioned patients
are presented in Table 3.
One can see that the content of uric acid in comparison with urea for all the
patients with pulmonary tuberculosis is higher than after a month’s treatment.
Some of the patients have a sufficiently higher value. After a 5-month’s treatment
this parameter decreases even more, as a rule.
5. THE ABSORPTION SPECTRA OF PULMONARY CONDENSATE OF
MAN
The results of spectrophotometric investigations of pulmonary condensate within
the interval 215    340 nm are presented in Fig. 5. One can see that in the
whole spectral interval the optical densities of pulmonary condensate of the
patients with pulmonary tuberculosis are sufficiently lower than those for the
healthy people. Taking into account the data, presented in literature [3,4], one can
state that the total content of BAS, including free amino acids in the pulmonary
condensate of the tuberculosis patients is sufficiently lower than that of the
healthy people. This is true for practically all cases (9 patients and 10 healthy
people). Similar conclusions obtained on the basis of biochemical analyses [3,4]
can be made as to the content of
hormones,
histamine
and
serotonin for the pre-asthma and
bronchial asthma patients.
As the tyrosine is not readily
soluble in water [7], it is
practically
unavailable
in
pulmonary condensate. So, let’s
consider the possibility of
quantitative determination of
phenylalanine and tryptophane
concentration in it, using the
analytical method of absorption
spectral analysis. Let’s use for
this
purpose
the
spectral
extinction
curves
for
the
mentioned amino acids [8], having chosen 1  265 nm as an analytical wavelength for phenylalanine and  2  285 nm – for tryptophane. The expressions for
calculating the correspponding concentrations C will be as follows:
Ct 
D 2
;
  2t l
Cf 
D1
 1 f l

D 2  1t
 1 f   2 t l
(4)
where l – the thickness of the pan (l=1 cm);  1 f ,  1t ,   2 f =0,   2t - extinction of
phenylalanine and tryptophane for the mentioned wave-lengths. The results of
calculations for a number of patients are presented in Table 4. As far as we can
see, the concentration of free amino acids for all the patients with pulmonary
tuberculosis is lower than that of the healthy people. Besides, the content of
phenylalanine is always higher than that of tryptophane.
Table 4
Concentration (mg%) of aromatic amino acids in pulmonary condensate
Healthy people
Amino acids
№3
№4
№1
Phenylalanine 0.708 0.597
1.82
0.56
0.486
0.41 0.310 0.06
Tryptophane 0.246 0.200
0.58
0.47
0.146
0.17 0.171 0.03
2.40
1.03
0.632
0.58 0.481 0.09
Total
№1
№2
Patients
0.954 0.797
№2
№3
№4
6. CONCLUSIONS
The results obtained prove that spectrophotometric technique of investigating the
optical properties of biological media (plasma, urine and pulmonary condensate of
man) can be used in the routine laboratory practice of antituberculosis
establishments as one of the ways of determining the activity of the process and
control of the efficiency of treating the patients with pulmonary tuberculosis.
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