Evaluation and modeling of kinetics of Aluminium in plasma and dialysis fluid.

S.C. Pun 1, M.S. Tudosie 1, 2, R. Macovei 1,3, Luminia Ardelean 1,3, Viorica Bumbea3, Genica
Caragea2, M. Ionic 2, C. Mircioiu1,2, A. Piperea-Sianu1, C. Mladin4
1
University of Medicine and Pharmacy Carol Davila Bucharest
2
Army Center for Medical Research, Bucharest
3
Clinical Emergency Hospital, Bucharest
4
Mecro System Bucharest
ABSTRACT
Paper investigated kinetics of Aluminium levels during renal dialysis. Plasma and dialysis
fluid levels of Aluminium where evaluated using an Atomic Absorption Spectrometry method.
Evaluation included determination of mean blood level of a control group and a chronic renal
failure patients group. It was found that plasma levels of Aluminium (21.7 22.5 g/L) were
significant higher in patients than in control group (4.07 0.51 g/L). Concentration of Aluminium
in dialysis fluid was two times lower than mean concentration in blood (8.78 8.90 before dialysis
and 9.0 13.2 g/L after dialysis). The difference between the mean concentrations at the
beginning and at the end of dialysis was not significant.
Consequently, the main source of Al seems to be the blood in the case of uraemic patients
and the dialysis work as a detoxification method.
In conditions of replacing tap water for dialysis with purified water obtained with reverse
osmosis the main Al flux is from blood to dialysis fluid.
Key words: Aluminium, pharmacokinetic model, haemodialysis, Atomic Absorption
Spectrometry, chronic renal disease
INTRODUCTION
Haemodialysis is a therapeutic technique that removes toxins from blood by allowing
equilibration of their plasma and dialysis fluid concentrations across a semi-permeable membrane.
In order to avoid significant modifications of serum concentrations of essential ions such as
potassium, sodium, bicarbonate, and calcium in blood, dialysis fluid contains these ions at
concentrations comparable with their physiological level.
Because the dialysis fluid concentration of other substances such as trace elements is not
routinely manipulated, substances that have lower concentrations in dialysis fluid than in blood tend
to be removed by dialysis. This phenomenon is appropriate in the case of uraemic toxins, but it may
lead to depletion of biologically essential substances.
In case of impaired renal function patients trace element disturbances are due to: reduced
renal function, proteinuria, alterations of gastrointestinal absorption and the dialysis procedure [24].
Haemodialysis procedure used very high volumes (>300 L/week) of dialysis fluid for one
patient. Therefore, even low levels of toxic substances in source water could lead to concentration
gradients between blood and dialysis fluid, which in turn could lead to blood contamination and
some substances present in dialysis fluid but not in blood will tend to accumulate in the patients
body, and the lack of renal clearance in haemodialysis patients might theoretically lead to toxicity.
Thus, the chronic renal failure patients are at theoretical risk for both deficiency and accumulation
of trace elements, depending on removal by dialysis, the composition of the source water used for
haemodialysis [1-6].
Many chemical elements existing in nature are also found in the human body, most of them
playing an important role. Homeostatic mechanisms keep their concentration in the human body
within a normal range. Their concentration in body fluids can be analytically evaluated. The
biological samples used to determine these concentrations are: blood, urine, cerebrospinal fluid,
hair, nails, tears and sweat [2, 7, 8].

Aluminium is not proved to have a physiological function in the human body. Digestive
absorption of Aluminium is less than 1 % of the daily ingested dose [8]. It concentrates in bones and
liver and has a renal elimination [8].
Aluminium sources are: contaminated water, food packs, protection films for microwaves,
vaccines and cosmetics. In chronic dialysis patients case, serum concentrations of Aluminium in
the past were significantly higher due to inadequate dialysis water [8].
Tap water is the main source of dialysis fluid aluminium contamination. In order to avoid
aluminium contamination, tap water should be treated by softening, deionization and reverse
osmosis [24].
Another source of aluminium is aluminium-containing medication. For patients who receive
such medication it is highly recommended serum aluminium testing every three months and for
uraemic patients not receiving aluminium-containing medication at least once a year [25].
Use of phosphorus chelators, containing Aluminium, has as result the accumulation of
aluminum. This condition has been called "dialysis dementia", "renal osteodystrophy with low turnover" or "osteomalacia" [8].
The major complication of aluminium intoxication in prolonged haemodialysis is
encephalopathy, first described in 1972, developing the following symptoms: dysarthria, dyspraxia,
myoclonic jerks and grand mal seizures. In terminal stages may occur facial grimacing, myotonic
spasm and dysphasia. Death is due to inanition or infection [24].
The treatment of patients overloaded with aluminium encounters a major problem giving the
fact that the aluminium disposal during haemodialysis is difficult due to the fact that plasma
aluminium ions are bound to high molecular weight plasma proteins, especially transferring [2628].
In order to discharge aluminium from the body patients must be treated with
desferrioxamine, a drug that mobilize aluminium from tissues [29] and plasma [30].
So, prevention of aluminium exposure is the main goal in the management of renal patients
in order to avoid aluminium overload [31].
The acceptable concentrations of aluminium in dialysis fluid are bellow 2 g/L [32].
The concentration of serum aluminium at the beginning of dialysis should be less than 20
g/L. Toxicity can occur at levels greater than 60 g/L [25].
Currently, the Aluminium concentration in dialysis fluid is below 10 g/L [8].
MATERIAL AND METHOD
Clinical protocol. The study was a randomized, cohort analysis for two groups of patients:
one group with normal renal function - control group in which were determined Aluminium blood
levels and one group of patients with chronic renal disease in which were determined Aluminium
blood levels, as well as time course of concentrations in dialysis fluid.
The inclusion criteria for the control group were: normal renal function and less than 65
years. The inclusion criteria for the second group were: chronic dialysis in a medical center, 13.5
hours of dialysis per week, age less than 65 years.
Control group included 50 subjects (26 % females and 74 % males), age 39.82 0.65 years.
They were considered health based on their creatinine normal values (0.93 0.22 mg/dL, range
0.56 - 1.5).
Patient group included 51 patients (35.3 % females and 64.7 % males), age 53.62 11.34
years.
Vascular approach was made by arterial-venous fistula or long - life catheter. Dialysis fluid
flow was 500 mL/min.
In the course of the study, the samples were taken as follows:
- control group 1 mL of blood in a tube with heparin for determining Aluminium, and
blood for determination of creatinine level;
- patients group - 1 mL of blood in a tube with heparin before the dialysis starts; 1 mL of
blood at the end of dialysis; 10 mL of dialysis fluid at time intervals fixed by the protocol study.

Bioanalytical method. For the evaluation of the studied elements, it was used a
spectrometry installation of atomic absorption spectrometer with Atomization in Graphite Furnace
GF-AAS [9], AAS Varian installation: SpectrAA 880 atomic absorption spectrometer; atomizer
with Graphite Furnace GTA 100; auto sampler PSD 25; water cooler CFT 33 Neslab; nitrogen
generator, analytical nitrogen 99.999 % purity; analytical argon 99.999 % purity (Linde).
Blood samples (1 mL) were collected in anticoagulant tubes at connecting and restitution
dialysis fluid.
The dialysis fluid samples (10 mL) were taken during the dialysis at 30 min, 1, 2, 3, 4 hours
from the time when dialysis started.
Blood processing method consisted in mixing 200 L blood with 800 L antifoam B and 1
ml 1.6N HNO3. After 20 minutes, the samples were centrifuged for 10 minutes at 2000 rot/min. 500
L supernatant for the injection in the installation. [10]
Dialysis fluid processing method consisted in addition of 1mL nitric acid 65 %. After 20
minutes, the sample was centrifuged for 10 minutes at 2500 rot/min. The supernatant represented
the matrix for the injection in the installation.
Determination of Aluminium levels included the following steps: automatic dilutions; 20
L injection.
Working parameters. To determine Al concentration from samples we used the following
method: injected sample 20 L; measuring method peak height; smoothing 9 pts; replicated
no. - 2 for standard and sample; cathode tube current 10 mA; wavelength 309.3 nm; split width
0.2 nm; background correction deuterium lamp; drying 120C; calcinations 1000C;
atomization 2700C; cleaning - 3000C; calibrating in concentration, 3 points calibration curve
0, 50, 100 g/L; recalibration rate -15. Automatically quality control of dispersion (<10 %);
correlation coefficient (> 0.998); DLM (< 0.5 g/L); DLI (< 0.05 g/L) [4].
A rational calibration curve [4] was stabilized to fit to model:
A / C a bA cA 2

A - absorbance of the sample;

C - concentration of the sample;
a, b, c calibration curve coefficient.
Correlation coefficient needed in automatically quality control was lower than 0.998.
Results and discussions
Mean value of plasma levels in control group was found 4.07 g/L with a variation
coefficient greater than 80 %. This value is in agreement with the mean value of approximately 5
g/L found in literature [23].
Plasma levels in the group of patients before dialysis was found 21.7 22.5 g/L (range
0.1 91). This value is situated between the value of plasma levels of uraemic patients not
consuming Al medications (11 g/L) and patients consuming Al (50 g/L) [23].

Fig. 1. Plasma levels of Aluminium before and after dialysis.

Mean plasma level after dialysis appeared to be lower than the value before (18.1 g/L face
to 21.7 g/L) but the difference, due to high variability of data, Fig. 1, couldnt be proved as
significant. The number of data was not enough great to check reliable if are or not normally
distributed. Supposing although that there are no reasons for suspicion lack of normality the
means were compared using Student test. Application of test gave a p value of 0.22, i.e. we cannot
reject the hypothesis that the values are equal. Some patients seem to have much higher values than
the rest of the group. Elimination of data which is different more than two standard deviations from
the mean as outlier didnt changed the statistical conclusion led to a lower difference between
values before and after dialysis. Previous published data [23] indicated a small increase of the
plasma level of Al after dialysis. Clinical trial was performed thirty years ago, when the necessity
of purification of water for dialysis was not yet discovered. Uncontrolled high content of Al tap
water was not unexpected. In our experiments water was purified by reverse osmosis and
comparison of results is not very reliable. High values of Al in our experiments are coming maybe
form ingestion of Al with food and drugs and not from dialysis water.
An evaluation, restricted only to the group of patients with small values of Al concentration
(< 25 g/L), was performed separately. In such conditions, the mean results became even much
closer.

Fig. 2. Correlation between concentrations before and after dialysis in case of patients with Al
concentration lowers than 25.
Correlation coefficient between the values before and after dialysis, Fig. 2, was high enough. It is
interesting to remark that variance of data around regression line is practically the same for all values. The
regression line had a slope of approximately 1, supporting the conclusion of lack of influence of dialysis on
the plasma levels of Al, if these values are low at the beginning of the experiment.
Time course of Al in dialysis fluid. Since Al was not found in water for dialysis but was measured in
plasma, it was expected a transfer from blood and its appearance in water. Time course of Al concentration
in dialysis fluid during a four hours interval is presented, for entire group of patients, Fig. 3.

Fig. 3. Time course of Aluminium concentration.

Similar to what happened in case of blood levels, patients look to be shared into two groups: one
greater normal group with Al concentrations in dialysis fluid lower than 10 g/L and a smaller group
with high values (between 10 and 70 g/L). Second group could be considered as a group of outliers or could
be considered as normal in context of high variability of data. Whatever the case is, it is underlined that
their elimination didnt change qualitatively the conclusions.
It is to note that mean concentrations in dialysis fluid are approximately on half of the concentrations
in blood. This suggests that, in case of water purified by reverse osmosis the quantity of Al in dialysis fluid
does not lead to a risk of Al intoxication. On the contrary, dialysis appears playing the role of a detoxification
method.
Since surely the comparison of clusters of curves is difficult, data corresponding to each measuring
time were replaced by their means. Representation of time course of mean concentrations is presented in
Fig. 4.

Fig. 4. Aluminium in liquid dialysate - mean values - time course.

An increase of concentration of Al in time is a possible interpretation of result but, since the
correlation coefficient is very small (r 2 = 0.13) hypothesis cannot be proved statistically.

Conclusions
1. Mean Aluminium concentration in patients was significantly higher in comparison with
control group,
2. Concentration of Al in dialysis fluid was some two times lower than mean concentration
in blood. Consequently, the main source of Al seems to be the blood in the case of uraemic patients
and the dialysis work as a detoxification method.
3. In conditions of replacing tap water for dialysis with purified water obtained with reverse
osmosis the main Al flux is from blood to dialysis fluid.
4. Although mean plasmatic Aluminium concentration was lower after dialysis, it was no
significant difference between mean plasmatic concentration after dialysis and the one before.
Consequently an epuration is maybe only a temporary, low effect.
This paper is partially supported by the Sectorial Operational Programme Human
Resources Development, financed from the European Social Fund and by the Romanian
Government under the contract number POSDRU/89/1.5/S/64153.
5

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