Effect of antioxidant taurine addition on the reliability ... · MCV Mean Corpuscular Volume MPV...

Al-Azhar university - Gaza

Deanship of Postgraduate Studies & scientific research

Biology Department

M.Sc. Program in Biological Sciences

Effect of antioxidant "taurine" addition on the

reliability of complete blood count (CBC) and red cell indices of whole blood� specimens stored at room

temperature and at 4 Co up to 7 days

Submitted in Partial fulfillment of requirements for the Degree of Master

of science (biological sciences)

Submitted By /Heba A. Al Sarraj

(B.Sc. in microbiology)

Supervisor Co-supervisor

Dr. Mahmoud Sirdah Dr. Abdel Nasser Abushahla

��November 2010��

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��DEDICATED��

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My great parents who love me, who raised me, supported me, and taught me everything I know.

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My brother Rajab and nephews. ��

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My sisters and niece. ��

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Acknowledgements��

My deepest and profound acknowledgments are to my supervisor Dr. Mahmoud

Sirdah for his continuous support, generous help, and fruitful and constructive

suggestions. Without his assistance and guidance this work could not be done. I

would like also to express my sincere gratitude to my Co- Supervisor Dr. Abdel

Nasser Abushahla for his support and constructive suggestions throughout this work.

My appreciation and thanks are extended to the staff of Biology department and the

staff of Biology laboratory in Faculty of Science at Al-Azhar university- Gaza, and

the stuff of Balsam laboratory for their help and support specially, Mr. Mohamed

Abdel Majeed.

My special deep and sincere gratitude goes to my parents, brother, sisters especially

Rana, and my friends for their continuous support, help, and encouragement.

Finally, thanks are extended to everyone who has helped in this work.��

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Table of Contents��

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Page

i��

ii

- Approval sheet .

- Dedication

iii iv��

- Acknowledgments ...

- Table of content

vii - List of Tables ....

ix - List of Figures....

x - List of Abbreviations ....

xi ��

xiii

- Abstract ....

- Abstract in Arabic..

1 I. Introduction ..

5 II. Review of Literature ...

5��

6

II.1. CBC and its hematological indice

II.2. Storage time and CBC.

7 II.3.Temperature and CBC .

9 II.4. The Anticoagulant EDTA ............

12 II.5. Taurine ..

12�� II.5.1. About Taurine

13 II.5.2.Taurine Biosynthesis ..

14 II.5.3. Actions of Taurine .

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14 II.5.3.1. Bile Acid Conjugation and Cholesterol Excretion .

15�� II.5.3.2. Detoxification By Taurine ...

17�� II.5.3.3. Membrane Stabilization of Taurine .

24 III. Materials and Methods ..

24 III.1. Study design.................

24 III.2 Target population .

24 III.3. Chemicals and equipments

24 III.4. Methods

24 III.4.1. Sampling .

26 III.4.2. Study Groups .

26 III.5. Statistical analysis .

28 IV.Results ..

28 IV.1. Baseline values of the samples ..

31 IV.2. Reliability of the CBC Parameters and Indices .

31 IV.2.1. Platelets Count (PLT) ..

36 IV.2.2. Mean Platelets Volume (MPV)

40 IV.2.3. Red cell distribution width (RDW) .

44 IV.2.4. Red blood cell count (RBC)

48 IV.2.5. Mean Corpuscular Volume (MCV) .

52 IV.2.6. Mean Corpuscular Hemoglobin (MCH) ..

56 IV.2.7. Mean Corpuscular Hemoglobin Concentration (MCHC)

vi

60 IV.2.8. Hematocrit Concentration (Hct) ..

64 IV.2.9. Hemoglobin concentration (Hb) ..

68 IV.2.10. White Blood Cell Count (WBC)

72 V.Discussion ...

73 V.1. Baseline values of the samples .

74 V.1.1. Platelets Count (PLT) .

75 V.1.2. Mean Platelets Volume (MPV) ..

76 V.1.3. Red Cell Distribution Width (RDW)

76 V.1.4. Red blood cell count (RBC) ..

77 V.1.5. Mean Corpuscular Volume (MCV) ...

78 V.1.6. Mean Corpuscular Hemoglobin (MCH)

78 V.1.7. Mean Corpuscular Hemoglobin Concentration (MCHC) ..

79�� V.1.8. Hematocrit Concentration (Hct)

79 V.1.9. Hemoglobin concentration (Hb)

80 V.1.10. White Blood Cell Count (WBC) .

81��

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83 84

V.I.Summary and Conclusion .

VI.1. Summary.

VI.2. Conclusions.........................................................................................

VII. Recommendations.

86��V.III.References

vii

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List of Tables��

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Page ��

��Table

29 Mean and SD of the baseline values (2-3 hours from collection) of CBC parameters of the blood samples at room temperature

Table1.

30 Mean and SD of the baseline values (2-3 hours from collection) of CBC parameters of the blood samples at 4 oC refrigerator

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Table 2.

33 PLT count for samples at Room Temperature ..

Table 3.��

34 PLT count for samples at Refrigerator . ��

Table 4.��

35 Mean Percentage changes of PLT ... ��

Table 5.��

37 MPV for samples at Room Temperature ...

Table 6.��

38 MPV for samples at Refrigerator . ��

Table 7.��

39 Mean Percentage changes of MPV . ��

Table 8.��

41 RDW for samples at Room Temperature . ��

Table 9.��

42 RDW for samples at Refrigerator .. ��

Table 10.��

43 Mean Percentage changes of RDW ��

Table 11.��

45 RBC count for samples at Room Temperature ��

Table 12.��

46 RBC count for samples at Refrigerator ��

Table 13.��

47 Mean Percentage changes of RBC .. ��

Table 14.��

49 MCV for samples at Room Temperature .. ��

Table 15.��

50 MCV for samples at Refrigerator . ��

Table 16.��

51 Mean Percentage changes of MCV . ��

Table 17.��

53 MCH for samples at Room Temperature . ��

Table 18.��

54 MCH for samples at Refrigerator . ��

Table 19.��

55 Mean Percentage changes of MCH .

Table 20.��

57

MCHC for samples at Room Temperature . Table 21.��

viii

58 MCHC for samples at Refrigerator .. ��

Table 22.��

59 Mean Percentage changes of MCHC .. ��

Table 23.��

61 Hct for samples at Room Temperature .. ��

Table 24.��

62 Hct for samples at Refrigerator ... ��

Table 25.��

63 Mean Percentage changes of Hct .. ��

Table 26.��

65 Hb concentration for samples at Room Temperature .. ��

Table 27.��

66 Hb concentration for samples at Refrigerator ... ��

Table 28.��

67 Mean Percentage changes of Hb ��

Table 29.��

69 WBC count for samples at Room Temperature ... ��

Table 30.��

70 WBC count for samples at Refrigerator .. ��

Table 31.��

71 Mean Percentage changes of WBC .

Table 32.��

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ix

List of Figures��

Page ��

��Figure

12 Structure of taurine

Figure 1.

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List of Abbreviations:��

Full word��Abbreviation 2,2'-Azobis(2-amidinopropane) dihydrochloride��AAPH��Hepatic alpha-Smooth Muscle Actin��alpha -SMA Complete Blood Count CBC��Carbon Tetrachloride��CCl4��Cysteine Sulfinic Acid Decarboxylase��CSAD��Differential Leucocyte Count��DLC��5,5-dimethylpyrroline-N-oxide��DMPO Ethylene Diamine Tetra Acetic Acid��EDTA Electron Paramagnetic Resonance��EPR Haemoglobin Hb Haematocrit Hct Hepatic Damage��HD Hypochlorous acid��HOCl��Hepatic stellate cells ��HSC��International Council for standardization in Haematology��ICSH��intraperitoneal��i.p.��Tripotassium Ethylene Diamine Tetra Acetic Acid��K3 EDTA��Low density Lipoprotein��LDL Low density Lipoprotein_Cholesterol��LDL-CHO��Lipid Peroxidation��LP Lipopolysaccharide��LPS��Mitogen Activated Protein kinase��MAP-kinase��Malondialdehyde��MDA��Mean Corpuscular Haemoglobin MCH Mean Corpuscular Haemoglobin Concentration MCHC Mean Corpuscular Volume MCV Mean Platelet Volume MPV National Committee for Clinical Laboratory Standards��NCCLS��8-hydroxy-2\- deoxyguanosine ��8-OHdG��Photoreceptor terminals ��P1��Platelets Count PLT Optical platelet count��PLTo��Red Blood Cell RBC Recommended Daily Allowance��RDA��Red Cell Distribution Width RDW Monochlorotaurine��TauNHCl��Taurocholic acid��TCA Total Cholesterol��T-CHO��Triglyceride ��TG��Thepatic cytokine transforming growth factor-beta��TGF-â1 mRNA��Tauroursodeoxycholic��TUDCA��United Kingdom��UK United States��USA Very Low density Lipoprotein��VLDL��Very Low density Lipoprotein_Cholesterol��VLDL-CHO��White Blood Cell WBC��

xi

Abstract

Complete blood count (CBC) and its associated hematological indices are one of the

most common and routine laboratory tests. the present study aimed at the evaluation

of possible effects of adding antioxidant taurine on reliability and stability of

complete blood count parameters and its related cell indices of whole blood stored in

vitro at room temperature and 4oC refrigerator up to 7 days.

Random venous blood samples (20 ml each) were collected in K3-EDTA tubes

from 25 apparently healthy nonsmoker male students. The collected blood was

distributed almost equally into 2 sets of K3-EDTA tubes, each set of 4 tubes. One set

of EDTA tubes was kept at the ambient room temperature (23 ± 2 oC) while the other

set was kept at 4oC. For each temperature set, the four EDTA tubes correspond to:

control with zero taurine, 2.5 g/l taurine, 5 g/l taurine, and 10 g/l taurine

concentrations. For each group, CBC analysis was performed at collection time and

then daily for 7 successive days after collection and storage. Statistical comparisons

of data were carried out using the paired t-test, and one-way analysis of variance

(ANOVA). In addition, Mean Percentage changes were calculated and compared.

PLT count was significantly affected by storage temperature and time. Storage of

EDTA blood with taurine at room temperature considerably enhanced the stability of

PLT count over 7 days of storage, while taurine at 4oC showed no remarkable effect

on the stability of PLT count. Neither storage temperature nor incubation with taurine

at the different concentrations stabilize MPV of blood samples, with mean percentage

change increased considerably per time and reached values of 25.4 and 28.1 % at

room temperature and at 4oC, respectively on the 7th day.

xii

RDW readings were stabilized with taurine addition at the different concentrations for

48 hours at room temperature, and for 72 hours with 5 g/l taurine at 4oC refrigerator.

RBC count showed instability over time at the different temperatures, while taurine at

10 g/l exerted some stabilizing effect on RBC at room temperature over the first 5

days of the storage period. The MCV readings were stable for 2 days both at room

temperature and at 4oC refrigerator, and taurine did not enhance the stability of MCV

neither at room temperature nor at 4oC refrigerator. On the other hand, MCH readings

were found to be unstable along the 7 days both at room temperature and at 4oC

refrigerator, but addition of taurine to these samples exerted some stability on the

MCH readings both at room temperature for 5 days and for 4 days at 4oC refrigerator.

MCHC readings were unstable and exhibited a significantly decreasing trend

with time despite of temperature or addition of taurine. Also, Hct values were

unstable and exhibited an increasing pattern at room temperature and at 4oC

refrigerator even in the presence of taurine. The values of Hb concentration showed

stable values over the 7 days at both temperatures, with no advantage of taurine on the

stability of Hb values. While WBC count revealed different stabilities at room

temperature and at 4oC refrigerator which lasted for 7 days and 4 days respectively.

However, at room temperature taurine at 5.0 and 10.0 g/l reduced the stability to 3 and

2 days, respectively.

It was concluded that: the different CBC parameters and its related indices

exhibited variable stability patterns in terms of the storage temperature, the time

period and the addition of taurine. Therefore, it is worthwhile for each laboratory to

consider what conditions to be adopted when preserving EDTA blood according to

what laboratory tests are of concern and intended to be performed.

Keywords: Taurine, antioxidant, CBC, K3-EDTA, ANOVA and PLT.

xiii

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�ΔϳϮΌϤϟ�ΔΒδϨϟ�ςγϮΘϣ��

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xiv

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I. Introduction

Complete blood count (CBC) and its associated hematological indices are one

of the most common and routine laboratory tests that are requested as the first step to

diagnose an illness or clinical presentation. With the development of the automated

hematological analyzers or counters, the CBC test has become an easy, quick, and

reliable test that can give valuable information to physicians leading to provisional

diagnosis or to further testing (England et al., 1984; Buttarello, 2004).

Most clinical laboratories are equipped now with modern automated analyzers

that are capable of processing large number of hematological tests in an efficient and

timely manner. These tests include CBC [red blood cell (RBC), haemoglobin (Hb),

haematocrit (Hct), white blood cell (WBC), and platelets (PLT) counts] and its

related indices [mean corpuscular volume (MCV), mean corpuscular haemoglobin

(MCH), mean corpuscular haemoglobin concentration (MCHC), red cell distribution

width (RDW), and mean platelet volum (MPV)].

To ensure reliability of the results generated by the instrument, it is imperative

that the specimens are collected appropriately in a suitable anticoagulant and analyzed

on a properly calibrated instrument within the time frame considered appropriate or

recommended by the manufacturer (International Council for standardization in

Haematology (ICSH), 1993; Lewis, 1999; Macey et al., 2002).

However, the recent trends towards large centralized laboratories, and changes

in laboratory organizations, have brought redistribution activity to a new perspective.

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Laboratories now test specimens that have been dispatched over a long distance; as a

result, testing is often delayed by 1224 hours or more after veinpuncture. Moreover,

on weekends, this interval may exceed 36 hours. Therefore, When such a specimen

arrives, the laboratory must decide whether to accept or reject the specimen (Gulati et

al., 2002). The laboratory must then decide whether to perform all of the tests ordered

or only those deemed appropriate given the age of the specimen, and what comments

should be appended to the reported results regarding the reliability or limitations of

the analysis. Although laboratories should still give reliable results, excessive delays

in processing might affect the reliability, accuracy and imprecision analysis

(Buttarello 2004).

Taurine has been demonstrated to function as a direct or indirect antioxidant.

Directly, taurine performs its antioxidant effect by scavenging or quenching reactive

oxygen species and other free radicals, especially for the hypochlorite ion (Wright et

al., 1985) and inhibiting lipid peroxidation by reducing the production of

malondialdehyde (MDA), an end-product of lipid peroxidation, from the unsaturated

membrane lipids (Huxtable, 1992). However, the indirect antioxidant effect of

taurine is thought to be due to its ability to stabilize biomembranes structure and

function by preventing the increase in membrane permeability from oxidants effects

(Wright et al., 1985).

A lot of studies about the effect of taurine as antioxidant in different tissues

have been performed and confirmed the potent antioxidant activity of taurine

(Nakashima et al., 1982; Milei et al., 1992). Taurine has been demonstrated to act as

�

both a primary antioxidant that scavenges free radicals and as a secondary antioxidant

that attenuates oxidant-induced changes in biomembranes stability (Koyama et al.,

1992). It seems likely that taurine is involved in membrane stabilization at several

levels and that this action accounts for many of its physiological effects. Taurine's

ability to stabilize cell membranes may be attributed to one or more of its action:

antioxidation (Nakamori et al., 1990; Vissers et al., 1994), osmoregulation (Ozasa

&. Gould 1982), and its ability to maintain of intracellular ions homeostasis (Qi, et

al., 1995), its ability to inhibit membrane proteins phosphorylation (Lombardini,

1985), its ability to prevent lipid peroxidation (Pasantes-Morales & Cruz, 1985),

and its ability to form conjugated compounds (Hino et al., 1993).

Different studies have revealed that whole blood samples become more stable

when stored at low temperature or when an anticoagulant is used in preserving the

collected whole blood samples (McShine et al., 1990; Chen et al., 1999; Macey et

al., 2002; Buttarello, 2004; Mahmoodi et al., 2006).

Objectives of the study

The general objective was to evaluate the possible effects of adding the

antioxidant taurine on the stability and reliability of complete blood count and red cell

indices of whole blood stored in vitro at room temperature and at 4 oC for up to 7

days.

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The specific objectives were

To assess the effect of storage temperature (4 oC and room temperature) on the

stability and reliability of CBC and red cell indices of whole blood.

To test the effect of taurine addition at different concentrations on the stability

and reliability of CBC and red cell indices of whole blood.

To find any relationship between taurine concentration and stability and

reliability of CBC and red cell indices of whole blood.

To determine the best running period for each test of interest that provides

stable and reliable results.

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II. Review of Literature

The main objective of any laboratory determination is to produce results that

are accurate and precise enough for clinical use (Buttarello, 2004).

In clinical laboratories, the cycles of each test starts with the preparation of

patient, and continues with biologic sample collection, preparation and storage of the

sample under suitable conditions and finally finishes by reporting the result. It is

sometimes necessary to repeat a test to obtain a reliable result. Sometimes there is a

period of time between sample collection and doing or repeating a test. In this

situation the stability of the sample is very important. Since blood tests are more

common than the other biologic fluids, therefore using the standard methods for

sample collection, and storage, and the role of environmental factors that may affect

the bloods indices should be considered (Mahmoodi et al., 2006).

II.1. CBC and its hematological indices

The complete blood count (CBC) with its indices is one of the most common

laboratory tests performed nowadays. It gives information about the production of all

blood cells and identifies the patient's oxygen-carrying capacity through the

evaluation of red blood cell indices, hemoglobin, and hematocrit. It also gives

information about the immune system through the evaluation of the differential white

blood cell count. These tests are useful in diagnosing anemia, certain cancers,

infection, acute hemorrhagic states, allergies, and immunodeficiencies as well as

monitoring the side effects of certain drugs that cause blood dyscrasias (George-Gay

and Parker, 2003).

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Clinical laboratories equipped with modern automated analyzers are capable

of processing large number of hematological tests in an efficient and timely manner.

These tests include CBC , RBC, Hb, Hct, WBC, and PLT and its related indices

MCV, MCH, MCHC, RDW, and MPV. To ensure reliability of the results generated

by the instrument, it is imperative that the specimens are collected appropriately in a

suitable anticoagulant and analyzed by a properly calibrated instrument within the

time frame considered appropriate or recommended by the manufacturer (ICSH,

1993; Lewis, 1999; Macey et al.; 2002).

II.2. Storage time and CBC

The recent trends towards large centralized laboratories, and changes in

laboratory organizations have brought redistribution activity to a new perspective. In

fact, delayed sample analysis is not rare in clinical or laboratory routine, especially

when blood samples are shipped, under poorly controlled conditions to distant

centralized laboratories (Lippi et al., 2005). So clinical laboratory professionals are

familiar with the scenario whereby a blood specimen is collected on a Friday but not

delivered to the laboratory for processing until the following Monday or even later.

When such a specimen arrives to the laboratory, the staff needs to decide whether to

accept or reject it; and if accepted, whether to perform all of the requested tests or

only those deemed appropriate based on the age of the specimen; and what

comments, if any, should be appended to the reported results regarding their reliability

or unreliability. Such decision making requires the lab. technician to be familiar with

changes known to occur in blood specimens during storage. The manufacturers of

automated analyzers and the published literature often cite that blood specimens kept

�

at either room temperature or at 4 oC (refrigerated) for up to 24 hours, generally yield

reliable results for complete blood cell count (CBC) (Gulati et al., 2002). However,

recent studies showed that specific information concerning the suitability, stability,

and reliability of whole blood specimens older than 1 day for various automated

hematologic tests are limited, particularly in the recent literature (Buttarello, 2004;

Hedberg & Lehto, 2009).

Mahmoodi et al., (2006) in their study entitled "survey of changes in

complete blood count and red cell indices of whole blood incubated in vitro at

different temperatures up to 48 hours" showed that delay in doing the CBC test can

lead to changes in some parameters, therefore the blood samples should not be left in

the laboratory and the test should be done on blood samples as soon as possible.

In Philadelphia Gulati et al., (2002) suggested that clinically reliable results

may be obtained for some CBC parameters, from specimens older than 1 day, when

analyzed on the Coulter Gen.S (Beckman Coulter, Miami, Fla). For example, Hb,

RBC, and MCH values are stable for samples of 7 days old, WBC value is stable for

samples of 3 days old, and platelets value is stable for samples of 4 days of storage at

room temperature, While WBC differential value is not stable or reliable for samples

of more than one day old.

II.3. Temperature and CBC

Different studies have showed that whole blood samples become more stable

when stored at low temperature or when an anticoagulant is used in preserving the

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collected whole blood samples (Vogelaar et al., 2002; Lippi et al., 2005; Hedberg &

Lehto, 2009).

In Italy Lippi et al., (2005) evaluated the stability of conventional and new

hematologic parameters in blood specimens stored for as long as 24 hours at 4°C. Of

the 21 hematologic parameters tested with the use of the Advia 120 hematologic

analyzer (Bayer Diagnostics), means for paired samples of specimens differed

significantly over the 24-hours storage period for Hct, MCV, percentage of

macrocytes, PLT count, MPV, reticulocyte count and percentage, and reticulocyte

hemoglobin content (all P

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of impedance platelet count yielded more reliable results than the routine PLTo. WBC

differential parameters, except eosinophils, were stable for up to 48 hours at +23 ± 2

oC. CBC parameters were stable for 72 hours, except MPV, which slightly increased

between 48 and 72 hours at +4 oC. WBC differentials were stable for 4872 hours,

with a slight decrease observed in absolute neutrophils and lymphocytes at +4 oC. The

authors concluded that CBC and differential parameters should be analyzed as soon as

possible after collection. If analysis is delayed for a longer period of time specimens

should preferably be maintained at +4 oC. The date and time collection should be

clearly indicated and, in cases where a delay in analysis does not enable accurate

results, the corresponding parameter should be omitted and substituted by a comment.

II.4. The anticoagulant EDTA

Ethylene Diamine Tetra Acetic Acid (EDTA) is the most commonly used

anticoagulant in evacuated tubes since 1950s. It inhibits the clotting process by

removing calcium from the blood. It has certain advantages over other anticoagulants;

it does not distort blood cells, making it ideal for hematology use. So EDTA is the

anticoagulant recommended for full blood cell counts and white blood cell differential

analysis by the NCCLS (National Committee for Clinical Laboratory Standards),

principally for its cell preservation properties (Macey et al., 2002).

Potassium EDTA is the most soluble salt among the three other EDTA salts

Na2EDTA, K2EDTA and K3EDTA (England et al., 1993). Tripotassium Ethylene

Diamine Tetra Acetic Acid (K3 EDTA) is dispensed as a liquid and thus causes a

slight dilution of the specimen. This salt also affects the red blood cell size at

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increased concentrations and on storage than the dipotassium salt (Hedberg and

lehto 2009).

Nowdays the International Council for Standardization in Hematology

recommends the dipotassium salt of EDTA as the anticoagulant for full blood counts .

In Europe and Japan, it is the preferred anticoagulant, whereas in the United States

(USA) and the United Kingdom (UK) the tripotassium salt of EDTA is more

commonly used. Under optimal conditions (appropriate anticoagulant concentration

and analysis within 14 hours after veinpuncture), the choice of dipotassium EDTA or

tripotassium EDTA have insignificant effect on the results of full blood cell counts

and white blood cell differential analyses (Macey et al., 2002).

In Netherlands McShine et al., (1990) observed that platelet counts in citrated

blood samples were lower than those in EDTA and highlighted the necessity to

present citrated samples mixed with dried EDTA when characterization or quality

control of blood and blood components is required, and they suggested that a small

population of platelets may form microaggregates within 1 to 4 hours in the blood

collected in citrate-containing anticoagulant- solutions. This leads to an erroneous

evaluation of the platelet count. Mixing these samples with dried EDTA before

platelet counting will ensure a correct platelet count, if an EDTA sample itself is not

available (McShine et al., 1990 ).

In China Chen et al., (1999) found that values of Hb, Hct, MCV and

lymphocyte percentage collected in Na2 EDTA tubes were significantly higher than

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those collected in K3 EDTA (P 0.05 for Hb and lymphocyte percentage, and P all

0.01 for others ), While values of MCHC collected in Na2 EDTA were significantly

lower than those collected in K3 EDTA tubes ( P 0.05). for underfilling for blood

sample, values of Hct and MCV with 2 ml blood volume (both P 0.01 ), while

values of MCHC with 2 ml blood volume were significantly higher than those with

5ml blood volume ( P < 0.01 ). When the collection blood volume was increased to

3.5 ml, there were no significant difference between values for 3.5 ml and 5 ml blood

volume (P all > 0.05). in the storage stability study, there was a significant sequential

increase of Hct and MCV between 1 hour, 8 hours and 12 hours (P < 0.05 and < 0.01,

respectively, for 8 hours, P all < 0.01 for 12 hours). There was also a significant

sequential decrease of neutrophil percentage between 1 hour and 4, 8, 12 hours'

storage at room temperature (P all

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Figure 1: Structure of Taurine

analytical goals based on the state of the art are acceptable while for bias this is

satisfactory only for some parameters.

II.5. Taurine

I.5.1. About taurine

Taurine was first isolated from ox bile in 1827 and was named (Gallen-

Asparagin). After that, it was named Bos taurus, after the ox (Bos taurus is the Latin

name of ox). In 1838, the term taurine first appeared in the literature . Although

taurine was discovered more than 184 years ago, it is just only in the past three

decades that the significance of taurine in human nutrition and disorders has really

been investigated and identified (Sirdah, 2006).

Initial interest in taurine was generated during both World Wars, when naval

doctors had administered taurine to their soldiers to enhance their night vision and

relieve fatigue. Taurine is believed to be an all-round medicine (Sirdah, 2006) .

Taurine (2-aminoethanesulfonic acid

NH2CH2CH2SO3H), is a naturally-occurring ß-

sulfonated amino acid, its molecular weight is

125.2 and has two acidic dissociation constants

(pKa) values (at 25°C) of 1.5 and 8.82. Along

with methionine, cystine and cysteine, taurine is a sulfur amino acid (Sirdah, 2006).

Taurine zwitterionic nature gives it high water solubility and low lipophilicity

(Huxtable, 1992).

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Taurine occurs in the body as a free molecule or in simple peptides and is not

incorporated into muscle proteins. Taurine, one of the lesser-known amino acids, it

has many important and beneficial effects on the human body. It has been implicated

in many physiological functions, pharmacological actions and pathological conditions

(Sirdah, 2006).

For a long time, taurine was considered a nonessential nutrient for humans.

Recently, it has become clear that taurine is a very important amino acid involved in a

large number of metabolic processes and become essential under certain

circumstances (conditionally essential amino acid). When it is given in nutritional or

therapeutic dose, taurine has not been consistently linked with any toxicity or severe

adverse effects, it is thought to be quite safe (Sirdah, 2006).

II.5.2.Taurine biosynthesis :

Taurine biosynthesis was outlined by Jacobsen and Smith (Jacobsen and

Smith, 1968). There are a number of possible pathways through which taurine may be

generated, and these include:

1) the oxidation of cysteine to cysteine sulfinic acid and subsequently to

cysteic acid, which is decarboxylated to taurine.

2) oxidation of cysteine to cysteine sulfinic acid with subsequent

decarboxylation to hypotaurine, which is oxidized to taurine. The rate limiting step is

the conversion of cysteine sulfinic acid to hypotaurine catalyzed by cysteine sulfinic

acid decarboxylase (CSAD). This is a pyridoxal phosphate requiring enzyme, and

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therefore vitamin B6 deficiency can lead to a reduction in endogenous taurine.

Generally humans have an inherent capacity to maintain endogenous taurine levels

within a wide normal range from dietary methionine and cysteine. Because of this no

Recommended Daily Allowance (RDA) has been established for taurine (Redmond

et al., 1998).

The physiologic normal plasma taurine concentration is in the range between

35 and 60 mol/L, with any excess being readily excreted via the kidneys in the urine

(Trautwein and Hayes, 1990). Normal whole blood taurine in humans ranges

between 160 and 320 mol/L (Trautwein and Hayes, 1990).

II.5.3. Actions of taurine

Taurine has a lot of important and beneficial effects on the human body. It has

been implicated in many physiological functions, pharmacological actions and

pathological conditions (Sirdah, 2006).

II.5.3.1. Bile acid conjugation and cholesterol excretion

Bile acids, primarily cholic acid and chenodeoxycholic� acid, result from

cholesterol metabolism in the liver and are involved in�emulsification and absorption

of lipids and fat-soluble vitamins. In order for�this to occur, bile acids must be bound

to either glycine or taurine, forming�bile salt conjugates. The conjugation of bile acids

by taurine results in� increased cholesterol solubility and excretion (Hardison and

Grundy, 1983). In Japan Mizushima et al., (1996) had investigated the effects of oral

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taurine supplementation on lipids and sympathetic nerve tone in healthy young men

on experimental high fat and cholesterol diets. They found that oral taurine

supplementation attenuated increases in Total Cholesterol (T-CHO), Low density

Lipoprotein Cholesterol (LDL-CHO) and Low density Lipoprotein (LDL) in healthy

men on high fat cholesterol diets but induced significant increases in very Low

density Lipoprotein Cholesterol (VLDL-CHO), very Low density Lipoprotein

(VLDL) and triglyceride (TG), which could be explained by a possible effect of

taurine on lipoprotein lipase. Significantly lower urinary norepinephrine excretion

observed by the taurine administration implies the suppression of the sympathetic

nervous system.

II.5.3.2. Detoxification by taurine

Taurine has been demonstrated to function as a direct or indirect antioxidant.

Directly, taurine performes its antioxidant effect by scavenging or quenching reactive

oxygen species and other free radicals, especially for the hypochlorite ion (Wright et

al., 1985) and inhibiting lipid peroxidation by reducing the production of

malondialdehyde (MDA), an end-product of lipid peroxidation, from the unsaturated

membrane lipids (Huxtable, 1992). However, the indirect antioxidant effect of

taurine is thought to be due to its ability to stabilize biomembranes structure and

function by preventing the increase in membrane permeability due to oxidantation

effects (Wright et al., 1985).

A lot of studies about the effect of taurine as antioxidant in different tissues

have been performed and these studies confirmed the potent antioxidant activity of

taurine.

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Nakashima et al., (1982) investigated the effect of taurine on hepatic lipid

peroxide level following carbon tetrachloride (CCl4) administration in rats. To

determine the therapeutic efficacy of taurine administration on liver injury, to CCl4

intoxicated rats (CCl4, 2 ml/kg, intraperitoneal (i.p.)), 3 ml of a 10% aqueous solution

of taurine was administered after CCl4 administration (at 12, 16, and 20 hours after),

and rats were sacrificed at 24 hours after CCl4 administration in order to estimate the

level of lipid peroxides in the liver. The decrease of hepatic lipid peroxidation induced

by CCl4 was noticed in the taurine-treated rats. Taurine had no effect on mixed

function oxidase activity in hepatic microsomes nor on hepatic antioxidant content in

the CCl4-intoxicated rats. Hepatic taurine content decreased significantly 12 and 24

hours after CCl4 administration, whereas oral administration of taurine to CCl4-

intoxicated rats was able to protect these rats from hepatic taurine depletion. These

results suggest that hepatic taurine may play a critical role on the protection of

hepatocytes against hepatotoxins such as CCl4 and the administration of taurine may

be useful in the treatment of hepatotoxin-induced liver injury.

Miyazaki et al., (2005) examined the effects of taurine on hepatic fibrogenesis

and in isolated hepatic stellate cells (HSC). Rats of the hepatic damage (HD) group

were administered carbon tetracholoride (CCl4) for 5 weeks and a subgroup received,

in addition, a 2% taurine containing diet for 6 weeks (HDT). The HSC were isolated

from normal rats and cultured for 4 days. They found that hepatic taurine

concentration was decreased in the HD group. This loss and the hepatic histological

damage and fibrosis, were reduced following taurine treatment. Furthermore, the

hepatic alpha-smooth muscle actin (alpha-SMA), lipid hydroperoxide and 8-hydroxy-

2\- deoxyguanosine (8-OHdG) levels in serum and liver, as well as hepatic cytokine

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transforming growth factor-beta1 (TGF-â1 mRNA) and hydroxyproline levels were

significantly increased in the HD group, and most of these parameters were

significantly reduced following taurine treatment. In contrast to the mitogen activated

protein kinase (MAP-kinase) and Akt expressions, which remained unchanged, the

lipid hydroperoxide and hydroxyproline concentrations, as well as (TGF- â1 mRNA)

levels were significantly reduced by taurine in activated HSC. So oral taurine

administration enhances hepatic taurine accumulation, reduces oxidative stress and

prevents progression of hepatic fibrosis in CCl4-induced HD rats, as well as inhibits

transformation of the HSC.

A striking finding about the antioxidant action of taurine was reported by

Anitha Nandhini et al., (2002) who investigated the effect of taurine on the

susceptibility of the aorta to lipid peroxidation and on the activities of enzymatic and

non-enzymatic antioxidants in animal model. The study concluded that: not only

taurine supplementation reduces lipid perodxaition but it also raised the activities of

the antioxidant enzymes significantly (Anitha Nandhini et al., 2002; Sirdah, 2006).

II.5.3.3. Membrane stabilization of taurine��

Taurine ability to stabilize cell membranes may be�attributed to several

mechanisms. Taurine has been shown to regulate osmotic�pressure in the cell,

maintain homeostasis of intracellular ions, inhibit�phosphorylation of membrane

proteins, and prevent lipid peroxidation. As an�osmotic regulator, it has been

suggested that taurine, along with glutamic acid, act as an instrument in the transport

of metabolically-generated water from the�brain (Van Gelder, 1990).

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The antioxidation effects of taurine was investigated by many investigators as

Nakamori K. et al., (1990) who investigated the effect of taurine in protecting

biomembrane attacked by hypochlorous acid (HOCl) using canine erythrocytes which

had been pre-treated with HOCl. In the treatment, most of the HOCl was consumed as

a result of its reaction with a number of electrophilic substances, such as free amino

groups (-NH2) in the membrane, whereas hemoglobin inside the cells was not

oxidized. The lysis of HOCl-treated erythrocytes was dependent on the concentration

of HOCl and on the storage time at 37 °C. Taurine inhibited the lysis at 37 °C in a

dose dependent manner. During the storage of HOCl-treated erythrocytes with

taurine, an appreciable amount of monochlorotaurine (TauNHCl) was detected in the

supernate. This suggests that taurine might remove the oxidized chlorine from HOCl-

treated erythrocytes, resulting in the production of TauNHCl. The effect of taurine on

the removal of Cl+� moiety was further examined using Sepharose gel with free amino

groups. Taurine removed Cl+ moiety from HOCl-treated Sepharose gel, and the yield

of TauNHCl depended on the concentration of taurine and the storage time. These

results indicate that taurine might inhibit the hemolysis by scavenging the oxidized

chlorine moiety from the HOCl-treated erythrocytes.

Milei et al., (1992) investigated the possible free-radical scavenging action of

taurine during coronary artery bypass grafting, twelve patients were randomly divided

into two equal groups. One to 3 hours before surgery, they received a rapid

intravenous infusion of either placebo (group 1) or taurine (5 gm) (group 2). During

surgery, biopsy samples were taken before ischemia (preischemic samples) and after

10 minutes of reperfusion (reperfusion samples). Lipoperoxidation was determined by

hydroperoxide-initiated chemiluminescence of heart homogenates, and myocardial

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cell damage was assessed by electron microscopy. The values for chemiluminescence

in preischemic and reperfusion samples from group 1 were 7500 +/- 1600 and 18,600

+/- 4600 cpm/mg of protein, respectively (p less than 0.03). This difference was not

observed in group 2 where the values were 10,050 +/- 2700 and 11,800 +/- 4200

cpm/mg of protein, for preischemic and reperfusion samples, respectively. The

number of severely damaged mitochondria (grades 3 and 4) in reperfusion samples

from group 1 increased significantly compared to preischemic samples (25 +/- 8% vs

12 +/- 3%, p less than 0.01). Conversely no differences were observed between the

number of severely damaged mitochondria in reperfusion and preischemic samples

from group 2 (8 +/- 3% vs 8 +/- 2%). The number of damaged and necrotic myocytes

increased in group 1 after reperfusion from 22 +/- 9% to 34 +/- 10% (p less than 0.03)

and from 10 +/- 7% to 26 +/- 20% (p = NS), respectively. No differences were

observed between reperfusion and preischemic samples in group 2. Treatment with

taurine seems to reduce lipoperoxidation and decrease cell damage at the time of

reperfusion.

The effect of taurine in protecting biomembrane against oxygen radicals was

investigated by Nakamura T. et al., (1993) using canine erythrocytes 2,2'-Azobis(2-

amidinopropane) dihydrochloride (AAPH), a water-soluble azo-compound, was used

as the oxygen-radical generator. Taurine suppressed erythrocyte hemolysis more

effectively than alpha-alanine, used for comparison. To clarify the relationship to the

lipid peroxidation, the amount of lipid peroxide was measured using liposomes

prepared with egg yolk lecithin. However, the peroxidation was not suppressed by

taurine. When intact erythrocytes were subjected to hemolysis by hyposmotic

solutions, taurine suppressed the osmotic hemolysis more effectively than alpha-

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alanine. These results suggest that taurine does not have an antioxidative effect like

vitamin E, but interacts with the biomembrane, and helps to protect it against damage

caused by AAPH.

The study of Mankovskaya et al., (2000) in Ukraine was undertaken to

elucidate the effects of taurine on lipid peroxidation (LP) intensity and membrane

Na+, K+-ATPase activity in a hypoxic rat model. It was shown that 3 intraperitoneal

(i.p.) injections of 200 mg/kg of taurine prevented hypoxia-induced lactate

accumulation and LP in brain, liver, and heart tissues and prevented the decrease of

Na+, K+-ATPase activity in the liver. It is suggested that the effect of taurine on LP

could be due to the taurine antiacidotic action as well as to its membrane stabilizing

activity.

The effect of taurine on chimpanzee spermatozoa placed under hyperosmotic

stress was studied by Ozasa and Gould, (1982). The lethal effect of osmotic stress on

spermatozoa was partially alleviated by treating the cells with 2 mM taurine, whereas

the viability of unstressed cells was not affected by this treatment. Ultrastructurally,

exposure of spermatozoa to osmotic stress was accompanied by an increase in cells

with disintegrated plasma and outer acrosomal membranes. Treatment of stressed

spermatozoa with taurine partially prevented this increase with a concomitant

enhancement of cell viability. These results suggest that taurine plays an

osmoregulatory role in chimpanzee spermatozoa, which possibly involves

stabilization of the cell membrane.

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Membrane disorganization and swelling was induced by exposing the isolated

frog rod outer segments to light (5000 lux) in Pasantes-Morales and Cruz, (1985)

experiment . An increase of about 50% on lipid peroxidation, measured by the extent

of malonaldehyde formation, accompanied the light-induced damage. Taurine and

hypotaurine (25 mM) prevented the increase in lipid peroxidation, and provided an

entire protection of rod outer segment structure.

The maintenance of intracellular ions homeostasis by taurine was investigated

by Yamauchi-Takihara et al., (1988). The study was on chick heart and was

designed to investigate whether oral pretreatment with taurine or taurine added

directly to the perfusate has any effect on calcium paradox-induced heart failure. In

both protocols, taurine significantly reduced the mechanical dysfunction resulting

from the calcium paradox. Taurine pretreatment partially inhibited the excess

accumulation of calcium in the myocardium that occurs upon calcium repletion, and

microscopy revealed almost normal structure. This protective effect of taurine was

accompanied by: (a) reduction of the gain of sodium content that occurs during

calcium depletion. (b) reduction of the late gain in calcium that occurs during calcium

repletion. It is proposed that taurine plays a role in the regulation of calcium

homeostasis and membrane stabilization.

Qi et al., (1995) investigated the interrelationship between taurine and

erythrocyte-membrane Na-K ATPase activity. A comparison was conducted to test

whether taurine or uric acid (a water-soluble scavenger of free radicals) prevents or

recovers the depletion in membrane ouabain-sensitive Na-K ATPase activity resulting

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from ozone exposure or cholesterol enrichment of the erythrocyte membrane. A

depletion of 44% and 27% in ouabain-sensitive Na-K ATPase activity was

respectively caused by ozone exposure and cholesterol enrichment. Taurine like uric

acid partially prevented the activity loss from ozone exposure and taurine at high

concentrations (from 1.5 to 4.5 mM) restored the depletion of erythrocyte-membrane

Na-K ATPase activity due to ozone exposure and prevented the depletion of the

enzyme activity due to cholesterol enrichment. In contrast, although the same high

concentrations were used, uric acid failed to show any of the above effects. These

results suggest that taurine acts (1.5-4.5 mM) polyvalently as an antioxidizing agent

and as a membrane stabilizer to maintain the functions of membrane Na-K ATPase.

Lombardini, (1985) showed that taurine (20 mM) stimulates ATP-dependent

calcium ion uptake by two fold in crude retinal homogenates. On the other hand, it

inhibits the phosphorylation of specific membrane proteins as shown by acrylamide

gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-

aminoethylhydrogen sulfate demonstrated similar effects whereas isethionic acid and

guanidinoethanesulfonate showed no effect. A photoreceptor terminals (P1) (P1)

subcellular fraction of the retinal membrane preparation that contains photoreceptor

cell synaptosomes has a higher specific activity for the uptake of calcium ions.

Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition

of 20 mM taurine. Taurine is not affect on retinal ATPase activities or on phosphatase

activity, thus suggesting that it directly affects a kinase system.

Hino et al., (1993) studied the effect of lipopolysaccharide (LPS) and/or bile

acids on rat erythrocyte membranes in vitro. Addition of LPS isolated from E. coli

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into the erythrocyte resulted in a decrease of membrane fluidity as determined by spin

labelling using electron paramagnetic resonance (EPR). This was accompanied by

membrane fragility. Hydroxyl radicals were generated from erythrocytes treated with

LPS by using 5,5-dimethylpyrroline-N-oxide (DMPO) spin trapping. However,

pretreatment of erythrocytes with taurine-conjugated bile acids was found to modify

the membrane response induced by LPS. Taurocholic acid (TCA) and

tauroursodeoxycholic acid (TUDCA) prevented the decrease of membrane fluidity

induced by LPS, and, as a result, the membrane integrity was maintained and no

significant changes were observed in the amount of hydroxyl radicals produced by

LPS addition. However, taurochenodeoxycholic acid (TCDCA) exhibited little

beneficial effect on the dynamic properties and the function of the erythrocyte

membranes, although the hydroxyl radical declined markedly in the erythrocytes.

Therefore, it is suggested that TCA and TUDCA have a protective effect against LPS-

induced membrane fragility by modulating membrane fluidity.

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III. Materials and Methods

III.1. Study design

The present work was performed according to the experimental research design,

where case-control groups were compared and statistically analyzed for significant

differences of the study variables.

III.2. Target population

The target population of the present work was apparently healthy nonsmoker male

students (18-20) years old at Al-Azhar University Gaza.

III.3. Chemicals and equipments

Vacationer 5ml Tri-potassium Ethylene diamine tetra acetic acid (K3-EDTA) tubes

purchased from DISPO company, Amman-Jordan., while Taurine (99.5 % purity) was

purchased from Sigma Aldrich USA. The measurements were undertaken using an

Abbot CELLDYN 1800 Sapphire Hematology Analyzer ( Abbott Diagnostics, Santa

Clara, CA, USA ).

III.4. Methods

III.4.1. Sampling:

After the formal consent and approval of the project by the Biology

Department Council and the Deanship of Postgraduate Studies and Scientific

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Research at Al-Azhar university-Gaza, the male students of the faculty of science at

Al-Azhar University-Gaza were informed and invited to participate in the study.

Twenty five students 18-20 years old were involved and all subjects were informed

about the objective and scope of the study. They accepted to participate and all

signed consent forms about the study providing their acceptance to be enrolled in the

venous blood withdrawal procedure .

Random venous blood samples (20 ml each) were collected in K3-EDTA tubes

from 25 apparently healthy nonsmoker male students at the faculty of science, Al-

Azhar university-Gaza. The collected blood was distributed almost equally into 2 sets

of K3-EDTA tubes. One set was kept at the ambient room temperature (23 ± 2 oC)

while the other set was kept at 4oC refrigerator. Each set contains four tubes. Each

tube represented a study group therefore four study groups (one control and 3

different taurine groups) were included in each set of the present study.

For each group, CBC analysis was performed and including the following

parameters and indices CBC (RBC, Hb, Hct, WBC, and PLT counts) and its related

indices (MPV, MCV, MCH, MCHC, and RDW).

CBC was performed at collection time and then daily for 7 successive days after

collection and storage. For convenient sampling, testing and retesting, 8-10 blood

samples were withdrawn in each session that extend to 7 days. This led to performing

448-560 CBC tests /8- 10 blood samples of each session.

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III.4.2. Study groups

The four groups of each set were as follows:

Group I: 2.5 ml of venous blood is collected in K3-EDTA tube, and incubated

with 50 µl of normal saline solution, no addition of taurine (control group) .

Group II: 2.5 ml of venous blood is collected in K3-EDTA tube, with the

addition of 50 µl of 2.5 g/l taurine solution. (to increase taurine concentration

to 5 folds of the normal plasma taurine concentration)

Group III: 2.5 ml of venous blood is collected in K3-EDTA tube, with the

addition of 50 µl of 5 g/l taurine solution. (to increase taurine concentration to

10 folds of the normal plasma taurine concentration).

Group IV: 2.5 ml of venous blood is collected in K3-EDTA tube, with the

addition of 50 µl of 10 g/l taurine solution. (to increase taurine concentration

to 20 folds of the normal plasma taurine concentration).

Where the physiologic normal plasma taurine concentration is in the range between

35 and 60 mol/L (Trautwein and Hayes, 1990).

III.5. Statistical analysis

The results of CBC were grouped, tabulated, encoded and statistically

analyzed using the Statistical Package for the Social Sciences (SPSS) version 13.

Statistical comparison of data was carried out using the paired t-test, and one-way

analysis of variance (ANOVA). Any 2-tailed p value less than 0.05 was considered to

be statistically significant. In addition, for each parameter and indices the mean

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percentage changes (mean value mean baseline value / mean baseline value x100)

was calculated and compared.

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IV. Results

IV.1. Baseline values of the samples

The present work included 25 blood samples which were withdrawn from

apparently healthy, never smoker, male university students, aging 18-20 years old.

The blood from samples were divided into eight EDTA tubes, four were kept at room

temperature and the other four were kept at 4oC refrigerator. For each temperature, the

four EDTA tubes correspond to: control with zero taurine, 2.5 g/l taurine, 5 g/l

taurine, and 10 g/l taurine concentrations. The mean and standard deviation for all

CBC parameters and indices are mentioned in Table 1 for those samples stored at

room temperature and in Table 2 for those samples stored at 4oC refrigerator.

According to the ANOVA statistical test, non of these CBC parameters and indices

showed any significant differences neither as compared to the control group nor

among the taurine groups, with p-value > 0.05.

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Table 1: Mean and SD of the baseline values (2-3 hours from collection) of CBC

parameters of the blood samples at room temperature**

Stored at Room temperature

Parameter Control

n= 25

2.5 g/l taurine

n= 25

5 g/l taurine

n=25

10 g/l taurine

n=25

mean SD mean SD mean SD mean SD

PLT(K�uL) 220.08 51.20 228.20 65.52 214.88 50.84 228.08 54.66

MPV(fL) 8.77 0.74 8.84 1.03 8.57 0.52 8.97 0.99

RDW (%) 13.87 1.46 13.93 1.47 13.95 1.60 13.94 1.43

RBC (M�uL) 5.04 0.70 5.04 0.66 5.01 0.66 5.05 0.67

MCV (fL) 75.10 9.65 74.94 9.52 74.63 9.54 74.80 9.78

MCH (pg) 27.61 4.06 27.48 3.92 27.59 3.95 27.42 4.07

MCHC(g�dL) 36.64 0.98 36.57 0.82 36.92 0.92 36.56 0.88

HCT (%) 37.23 2.19 37.19 2.23 36.84 2.50 37.24 2.16

HB (g�dL) 13.65 0.98 13.60 0.94 13.66 1.04 13.62 0.96

WBC (K�uL) 5.55 1.44 5.49 1.33 5.52 1.33 5.53 1.31

PLT: platelets counts, MPV: mean platelet volum, RDW: red cell distribution width, RBC:

red blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular haemoglobin,

MCHC: mean corpuscular haemoglobin concentration, Hct: haematocrit, Hb: haemoglobin,

WBC: white blood cell.

** Non of these CBC parameters and indices showed any significant differences neither as

compared to the control group nor among the taurine groups, with P-value > 0.05.

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Table 2 : Mean and SD of the baseline values (2-3 hours from collection) of

CBC parameters of the blood samples at 4 oC refrigerator **

Stored at 4 oC Refrigerator

Parameter Control

n= 25

2.5 g/l taurine

n= 25

5 g/l taurine

n=25

10 g/l taurine

n=25

mean SD mean SD mean SD mean SD

PLT (K�uL) 222.96 57.16 229.48 62.09 229.48 62.09 236.12 51.70

MPV (fL) 8.81 0.59 8.81 0.70 8.81 0.70 8.89 0.70

RDW (%) 13.68 1.40 13.82 1.79 13.82 1.79 13.56 1.48

RBC (M�uL) 5.05 0.75 5.10 0.74 5.10 0.74 5.07 0.75

MCV (fL) 75.23 9.85 74.41 9.93 74.41 9.93 74.69 9.70

MCH (pg) 27.69 4.24 27.43 4.17 27.43 4.17 27.53 4.08

MCHC(g�dL) 36.70 1.08 36.75 0.97 36.75 0.97 36.75 1.00

HCT (%) 37.34 2.66 37.30 2.41 37.30 2.410 37.26 2.61

HB (g�dL) 13.70 1.03 13.70 0.96 13.70 0.96 13.69 1.00

WBC (K�uL) 5.73 1.43 5.59 1.40 5.59 1.40 5.63 1.36

PLT: platelets counts, MPV: mean platelet volum, RDW: red cell distribution width, RBC:

red blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular haemoglobin,

MCHC: mean corpuscular haemoglobin concentration, Hct: haematocrit, Hb: haemoglobin,

WBC: white blood cell.

** Non of these CBC parameters and indices showed any significant differences neither as

compared to the control group nor among the taurine groups, with P-value > 0.05.

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IV.2. Reliability of the CBC parameters and indices

In the following sections, the effect of the different storage temperatures

(Room temperature and 4 oC refrigerator) and the storage of the EDTA blood samples

with (2.5, 5, and 10 g/l) taurine on the different CBC parameters and indices are

presented. The reliability of the different CBC parameters and indices were evaluated

in term of the stability of these parameters and indices from the baseline values. Non

significant differences or changes from the baseline values indicated a stable and so

reliable parameter or indices. The mean ± SD for all parameters and were presented

and compared to the baseline corresponding values. Moreover, for each parameter the

mean percentage changes was calculated at the different storage temperatures (Room

temperature and 4oC refrigerator) and the storage of the EDTA blood samples with

(2.5, 5, and 10 g/l) taurine along the 7 days. The mean percentage change could be

used to describe the level of precision of the reading as compared to baseline values.

So when a precision level of 5 % is acceptable this reflect a mean percentage change

of ± 5 %.

IV.2.1. Platelets count (PLT)

Tables 3 and 4 show the effect of storage temperature, storage time, and the

storage with taurine at different concentrations on the stability of platelets count.

Platelets count was significantly changed and affected by the storage temperature and

storage time. However, the storage of the EDTA blood with taurine at room

temperature considerably enhanced the stability of PLT count over the 7 days of

storage and there were no significant differences in PLT count from the 1st day values

and the 7 days of storage at room temperature. While, the storage of the EDTA blood

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with taurine at 4 oC refrigerator showed no remarkable effect toward the stability of

PLT count, with some dispersed trend for storage with 5 g/l taurine. The mean

percentage changes in PLT count at the different temperature and taurine

concentrations are mentioned in Table 5. Except for the 7th day at room temperature

(11.8 %), all the values of mean percentage changes in PLT count were within ±10%.

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Table 3: PLT count for samples at room temperature

Parameter Day1 Day 2 Day 3 Day 4 Day5 Day 6 Day 7

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

control 220.08 51.20 235.68 57.79 237.00 55.49 233.64 54.08 232.48 56.92 225.04 60.13 244.20 54.78

2.5 g/l taurine 228.20 65.52 236.96* 66.98 240.48* 76.92 232.68* 72.38 227.24* 79.36 227.64* 72.99 240.64* 68.91

5 g/l taurine 214.88 50.84 231.80 58.19 228.16* 63.31 223.60* 65.79 217.96* 67.74 214.52* 62.83 228.00* 69.92

10 g/l taurine 228.08 54.66 230.52* 65.06 233.84* 64.16 234.12* 66.97 227.96* 62.23 220.44* 64.01 241.32* 69.53

* No significant changes were reported as compared to 1st day value.

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Table 4: PLT count for samples at refrigerator



control 222.96 57.16 232.28 60.60 226.52 54.36 243.20 62.18 234.76 65.07 243.44 70.86 228.12 59.73

2.5 g/l taurine 225.76 60.36 231.32 57.28 238.20 61.68 235.16 59.34 233.16 61.81 240.68 67.47 222.32 60.85

5 g/l taurine 229.48 62.09 229.52* 58.14 232.76* 56.86 241.04 62.34 234.40* 58.51 249.48 72.92 220.32* 63.28

10 g/l taurine

236.12 51.70 244.92 48.95 244.68 48.00 248.84 47.67 243.80 46.40 246.92 50.89 228.08 55.57


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Table 5: Mean percentage changes of PLT count compared to the 1st day values

Room temperature 4oC

Day2 Day3 Day4 Day5 Day6 Day7 Day2 Day3 Day4 Day5 Day6 Day7

PLT0 6.8 7.6 6.3 5.6 1.6 11.8 4.2 2.3 9.5 4.8 8.4 2.5

PLT2.5 4.3 5.8 2.3 -0.4 0.4 7.6 3.2 5.8�� 4.7�� 3.4�� 6.4 -1.5��

PLT5 8.4 6.8 4.5 1.4 0.3 6.7 0.7 2.3 5.6 2.8 8.3 -4.0

PLT10 1.0 3.2 2.8 1.3 -3.0 6.8 4.3 4.3 6.2 4.1 5.1 -3.7

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IV.2.2. Mean platelets volume (MPV)

Tables 6 and 7 showed the effect of storage temperature, storage time, and the

storage with taurine at different concentrations on the stability of mean platelets

volume (MPV). Neither the storage temperature nor the storage with taurine at the

different concentrations stabilize the MPV of the blood samples. The ANOVA

statistical test showed significant changes of MPV along the 7 days of storage, with a

trend towards an increase in the MPV of these samples. The mean percentage

changes in MPV at the different temperature and taurine concentrations are presented

in Table 8. The mean percentage change in MPV increased considerably per time and

at the 7th day it reached values of 25.4 and 28.1 % at room temperature and at 4oC

refrigerator, respectively.

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Table 6: MPV** for samples at room temperature



control 8.77 0.74 9.31 0.81 9.77 0.88 9.84 0.74 10.28 0.93 10.79 1.36 10.96 1.32

2.5 g/l taurine

8.84 1.03 9.23 0.66 9.72 0.49 9.91 0.72 10.18 0.84 10.41 1.64 10.45 1.18

5 g/l taurine

8.57 0.52 9.19 0.72 9.66 0.69 10.17 0.80 10.67 1.21 11.12 1.51 10.53 0.54

10 g/l taurine

8.97 0.99 9.53 0.61 9.81 1.01 9.81 0.62 10.86 2.02 10.43 0.92 11.51 2.09

** All readings are significantly different from the corresponding 1st day value.

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Table 7: MPV** for samples at refrigerator



control 8.81 0.59 9.44 0.70 9.90 0.85 10.16 1.04 10.58 1.18 11.11 1.57 11.33 1.10

2.5 g/l taurine

8.77 0.73 9.53 0.81 10.12 0.83 10.19 0.72 10.47 0.98 10.66 1.33 10.53 0.93

5 g/l taurine

8.81 0.70 9.36 0.77 10.07 0.84 9.73 0.58 10.44 0.84 10.73 1.02 11.09 0.73

10 g/l taurine 8.89 0.70 9.34 0.52 9.83 0.68 10.10 0.73 10.68 1.10 10.73 1.65 11.14 1.06

** All readings are significantly different from the corresponding 1st day value.

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Table 8: Mean percentage changes of MPV compared to the 1st day values



MPV0 6.2 13.1 13.9 19.1 24.5 25.4 7.1 12.3 14.5 19.7 25.7 28.1

MPV2.5 7.6 13.4 15.6 18.9 20.8 24.8 8.9�� 15.7 16.6 20.0 21.5 20.4

MPV5 7.3 12.7 18.7 24.3 29.3 23.1 6.3 14.3 10.7 18.7 21.8 27.2

MPV10 9.5 12.4 12.6 24.0 19.9 31.6 5.4 10.9 13.8 20.4 20.5 25.8

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IV.2.3. Red cell distribution width (RDW)

The possible effects of storage temperature and storage with taurine on the

RDW are illustrated in Tables 9 and 10. Table 9 showed that at room temperature the

RDW changed significantly over time stating from the second day readings. However,

the storage with taurine at the different concentrations stabilized the RDW readings

for the 48 hours from collection. While after that taurine has no stabilizing effect on

RDW at Room temperature, and all the RDW readings after the second day are

significantly differ from the baseline values.

At 4 oC refrigerator (Table 10), the RDW value is stable for 48 hours with and without

the effect of taurine. However, the storage with taurine at 5g/l enhanced the stability

of RDW value for additional 24 hours making it non-significantly changed from the

baseline value for 72 hours. On the other hand, the mean percentage changes on the

RDW are presented in Table 11, which revealed less than 10 % changes at room

temperature for the 1st 3 days, and less than 10 % changes at 4oC refrigerator for 5

days.

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Table 9: RDW for samples at room temperature



control 13.87 1.46 14.33 1.60 14.75 1.42 15.62 1.70 15.93 1.28 15.49 1.28 15.53 1.43

2.5 g/l taurine

13.93 1.47 14.06* 1.70 15.25 1.40 15.66 1.27 15.20 1.73 15.29 1.57 15.32 1.55

5 g/l taurine

13.95 1.60 14.12* 1.49 15.07 1.16 15.68 1.44 15.47 1.24 15.54 1.34 15.38 1.43

10 g/l taurine

13.94 1.43 13.91* 1.71 15.13 1.60 15.69 1.55 15.54 1.27 15.41 1.34 15.63 0.98


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Table 10: RDW for samples at refrigerator



control 13.68 1.40 13.83* 1.52 14.26 1.60 14.52 1.29 14.91 1.15 15.37 1.34 15.98 0.85

2.5 g/l taurine 13.81 1.42 13.77* 1.61 14.26 1.42 14.51 1.29 14.83 1.15 15.39 1.29 15.62 1.01

5 g/l taurine 13.82 1.79 13.52* 1.30 13.88* 1.35 14.36 1.40 14.93 1.25 15.54 1.48 15.63 1.32

10 g/l taurine 13.56 1.48 13.78* 1.75 14.22 1.32 14.35 1.37 15.00 1.27 15.63 1.10 15.70 1.17


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Table 11: Mean percentage changes of RDW compared to the 1st day values



RDW0 3.4 6.5 12.7 15.2 12.1 12.3 1.2 4.3 6.3 9.4 12.7 17.6

RDW2.5 0.9 9.7 12.8 9.2 10.0 10.1 -0.4 3.4 5.2 7.8 11.8 13.8

RDW5

1.3 8.5 12.9 11.5 11.9 10.6 -1.7 0.9 4.5 9.0 13.3 13.8

RDW10 -0.1 8.7 12.8 11.8 10.8 12.7 1.6 5.2 6.2 11.2 15.9 16.5

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IV.2.4. Red blood cell count (RBC)

Tables 12 and 13 show the effects of the different factors (storage time,

temperature, and addition of taurine) on the red blood cell count of the study samples.

Table 12 showed the instability of the RBC over time and the values were

significantly different from the baseline( p-value 0.05). Furthermore, Table 14, showed that the mean percentage changes in

RBC count is almost 3 % and 2.2 % at room and 4 oC refrigerator temperatures at the

7th day, respectively. While the addition of 2.5 g/l taurine reduces these mean

percentage changes to 1.6 and 1.2%, respectively.

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Table 12: RBC count for samples at room temperature



control 5.04 0.70 5.13 0.70 5.13 0.73 5.13 0.72 5.13 0.72 5.06 0.76 5.19 0.72

2.5 g/l taurine

5.04 0.66 5.09 0.67 5.14 0.68 5.08 0.70 5.09 0.73 4.99 0.72 5.12 0.71

5 g/l taurine

5.01 0.66 5.10 0.69 5.14 0.72 5.13 0.71 5.11 0.75 5.01 0.72 5.15 0.72

10 g/l taurine

5.05 0.67 5.09* 0.72 5.12 0.72 5.09* 0.74 5.06* 0.71 4.96 0.71 5.15 0.74


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Table 13: RBC count for samples at refrigerator



control 5.05 0.75 5.10 0.74 5.13 0.75 5.13 0.75 5.16 0.77 5.08 0.81 5.16 0.77

2.5 g/l taurine

5.11 0.76 5.16 0.73 5.16 0.72 5.19 0.72 5.19 0.73 5.09 0.75 5.17 0.77

5 g/l taurine

5.10 0.74 5.17 0.73 5.16 0.70 5.22 0.73 5.23 0.71 5.10 0.75 5.20 0.73

10 g/l taurine 5.07 0.75 5.16 0.73 5.17 0.72 5.21 0.73 5.21 0.73 5.05 0.71 5.17 0.74


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Table 14: Mean percentage changes of RBC compared to the 1st day values



RBC0 1.9 1.9 1.9 1.9 0.3 3.0 1.1 1.6 1.7 2.1 0.4 2.2

RBC2.5 1.1 2.1 0.8 0.9 -1.1 1.6 1.2 1.3 1.8 1.8 -0.2 1.2

RBC5 1.9 2.6 2.4 1.9 -0.1 2.9 1.5 1.5 2.5 2.8 0.1 2.1

RBC10 0.7 1.3 0.6 0.0 -1.9 1.8 1.8 2.1 2.9 2.8 -0.4 2.1

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IV.2.5. Mean corpuscular volume (MCV)

The stability of mean corpuscular volume (MCV) over the 7 days is summarized in

Table 15 for those samples kept at room temperature and in Table 16 for samples kept

at 4oC refrigerator. At all the investigated concentrations, taurine did not enhance the

stability of MCV neither at room temperature nor at 4oC refrigerator. However, MCV

reading itself was stable for 2 days both at room temperature and at 4oC refrigerator,

with no significant differences between the 1st and 2nd days readings. Moreover, the

mean percentage changes of MCV values (Table 17) were found to be increased by

time but these changes were lower when samples are at 4oC refrigerator, with value of

almost 10 % at day 7.

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Table 15: MCV for samples at room temperature



control 75.10 9.65 76.36* 10.05 78.00 10.06 80.93 10.51 83.42 11.29 84.58 11.43 85.85 11.78

2.5 g/l taurine 74.94 9.52 75.62 9.49 77.92 10.03 81.13 10.52 83.29 11.03 84.54 11.34 85.64 11.69

5 g/l taurine 74.63 9.54 75.62 9.51 78.02 9.88 81.12 10.50 83.48 11.04 84.87 11.48 85.78 11.82

10 g/l taurine 74.80 9.78 76.21 9.89 78.02 9.91 81.12 10.62 83.45 11.19 84.76 11.48 85.66 11.61


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Table 16 : MCV for samples at refrigerator



control 75.23 9.85 75.28 10.11 76.00 10.01 78.97 11.29 78.17 10.14 80.62 10.42 82.82 10.93

2.5 g/l taurine

74.30 9.99 74.97 9.58 75.35 9.88 76.65 9.98 78.02 9.99 80.36 10.27 82.44 10.52

5 g/l taurine

74.41 9.93 74.50 9.35 75.49 9.86 76.52 10.06 77.90 10.00 80.32 10.20 82.25 10.56

10 g/l taurine 74.69 9.70 74.54 9.58 75.24 9.71 76.40 9.90 77.88 10.21 80.37 10.51 82.38 10.79


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Table 17: Mean percentage changes of MCV compared to 1st day values



MCV0 1.7 3.9 7.7 11.0 12.5 14.2 0.0 1.0 5.2 3.9 7.2 10.1

MCV2.5 0.9 4.0 8.2 11.1 12.7 14.1 1.0 1.5 3.3 5.1 8.3 11.1

MCV5 1.4 4.6 8.7 11.8 13.6 14.8 0.2 1.5 2.9 4.8 8.1 10.6

MCV10 1.9 4.4 8.4 11.5 13.2 14.4 -0.2 0.8 2.3 4.3 7.6 10.3

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IV.2.6. Mean corpuscular hemoglobin (MCH)

Mean corpuscular hemoglobin readings were found to be unstable along the

7days both at room temperature and at 4oC refrigerator, (Tables 18 and 19,

respectively). But, the addition of taurine to these samples was found to exert some

stability on the MCH readings both at room temperature and at 4oC refrigerator. At

room temperature storage EDTA blood with 10 g/l taurine enhances the stability of

MCH over 5 days f

Effect of antioxidant taurine addition on the reliability ... · MCV Mean Corpuscular Volume MPV...

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