AnalyticalMethods

PAPER

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aInstitute of Genetics and Biochemistry, Fede

MG, Brazil. E-mail: nspenha@ufu.br; Fax: +

2203 ext. 23bFaculty of Medicine, Federal University of U

Cite this: Anal. Methods, 2014, 6, 1377

Received 16th August 2013Accepted 16th December 2013

DOI: 10.1039/c3ay41404c

www.rsc.org/methods

This journal is © The Royal Society of C

Kinetics of hypotonic lysis of human erythrocytes

Lucas Moreira Cunha,a Morun Bernardino-Neto,a Mario da Silva Garrote-Filho,a

Carla Braga Avelar,b Mariana Vaini de Freitas,a Rita de Cassia Mascarenhas Netto,a

Lara Ferreira Paraiso,a Letıcia Ramos de Arvelos,a Ana Flavia Mayrink Gonçalves-e-Oliveiraa and Nilson Penha-Silva*a

The curve of osmotic stability of erythrocytes is based on the amount of lysis as a function of salt

concentration under fixed time incubation and represents an equilibrium situation after a sufficiently

long time, although lysis is a rapid process. The curve is valid for the analysis of modulating agents that

have influence on this equilibrium, but not for those that have influence on the lysis kinetics. This work

has developed experimental conditions to study the hemolysis kinetics based on the interruption of lysis

by hypertonicity at predetermined intervals of time. These conditions were used to evaluate the kinetics

of hemolysis of 17 volunteers. The lysis curve as a function of time was statistically fitted to a hyperbola,

using the analytical routine of the integrated kinetic model of Michaelis–Menten to determine the time

required to promote lysis of half of the population of erythrocytes (t1/2) and the maximum absorbance

(Amax) reached in the test. The results showed good variance among volunteers. The constant t1/2 was

negatively correlated with total and LDL-cholesterol, and Amax, as it was expected, showed significant

associations with hematological variables that are under the influence of hemoglobin levels. Stratification

of the study population into two age groups (18–30 and 40–90 years old) showed that the t1/2 values

were significantly lower in the older population. Although the study population has been small, the study

showed that this kinetic approach of the erythrocyte lysis is very promising for analyzing the myriad of

variables which have influence on the cell membrane.

Introduction

To perform their functions adequately, biological membranesmust maintain their organizational structure and preserve theirphysicochemical properties.

Various agents and adverse conditions can affect the integ-rity of the membranes, thus causing damage to the functionsthey perform. However, the membranes are able to withstandcertain levels of agents or processes that can promote theirdestabilization. The ability of the membrane to maintain itsorganizational structure before being affected by these agents oradverse conditions is called stability.1

Factors that may affect the stability of the biologicalmembranes are basically similar to those that affect the stabilityof proteins, such as pH, temperature, drugs, destabilizing andstabilizing solutes.2 More specic factors, such as the relativeconcentration of the constituents of the membrane itself, mayalso cause changes in the stability of biological membranes.3

The erythrocyte is a widely used model to study membranes,because it is devoid of nucleus and organelles4 and also because

ral University of Uberlandia, Uberlandia,

55 34 3218 2203 ext. 24; Tel: +55 34 3218

berlandia, Uberlandia, MG, Brazil

hemistry 2014

it represents a practical model of study, since its lysis releaseshemoglobin, which can be easily quantied by spectropho-tometry in the visible region of the spectrum, and its acquisitionuses a minimally invasive procedure to the donor.

One way to analyze the stability of the erythrocyte membraneis in solutions with decreasing concentrations of NaCl (eryth-rocyte osmotic fragility). The prole of this analysis generates asigmoidal regression curve from which one can evaluate thestability of cells.1,5–8

This method, as well as others used in the study of thestability of biological membranes, is based on the principles ofthermodynamics, and consists of determining the variation of aproperty of the systemwhen it passes from one equilibrium stateto another. Normally to achieve this condition, a sufficientlylarge xed time interval is used to ensure that the process whichis under investigation has already occurred in its totality. If thevariable time is introduced in this analysis, the physicochemicalapproach of the study becomes kinetic. The kinetic analysis oflysis can distinguish the inuences of factors whose effects havedifferent velocities of action on hemolysis, although underincubation conditions by xed time these effects are equivalent.

The use of this kinetic approach may allow a better assess-ment of the impact of known factors, such as lipid composition,volume variability,9,10 deformability11 and stability5,12 on thehealth of the individual, once those variables are related to

Anal. Methods, 2014, 6, 1377–1383 | 1377

Analytical Methods Paper

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obesity,12 hypertension,13 diabetes,14 anemia15 and cardiovas-cular disease.9,10

The kinetics of biological processes more dened andextensively studied is the one of enzymes. The kinetics of enzy-matic reactions is usually based on the Michaelis–Mentenequation,16 which represents the hyperbolic dependence of theinitial reaction rate with increasing substrate concentration. Theuse of a non-linear hyperbolic statistical adjustment allows thedetermination of the maximal velocity (Vmax) that the enzymecan achieve in the state of quasi equilibrium (steady state), inwhich the substrate is present at saturating concentrations forthe catalyst, as well as the half transition point of the curve, fromwhich is obtained the constant KM, which represents thesubstrate concentration at which the enzyme reaches half of itsVmax. As the constants KM and Vmax are very signicant for thecharacterization of enzymes, many mathematical treatmentshave been developed to determine their values.

The mathematical treatments used in those classicalmethods for studying enzyme kinetics have been used in theanalysis of various biological and physiological processes. Thiswork uses the hyperbolic non-linear tting routine to analyzethe kinetics of lysis of erythrocytes.

Materials and methodsStudy population

The study was previously approved by the Ethics Committee ofthe Federal University of Uberlandia (Protocol 127/11). Thevolunteers were healthy people aged 18 to 90 years (n¼ 17), withno history of chronic consumption of alcohol and drugs.

Collection of blood samples

Blood samples were collected aer overnight (8–12 h) fasting, byintravenous puncture, directly into evacuated tubes containing0.1 g dL�1 K3EDTA as an anticoagulant (Vacutainer, BectonDickinson, Juiz de Fora, MG, Brazil).

Reagents and equipment

The NaCl used (Labsynth Diadema, SP, Brazil) had an ACSquality, with a purity of 99.5%, which was duly corrected forpreparing solutions. Volume measurements were always madewith the help of automatic pipettes (Labsystems, model Finn-pipette Digital, Helsinki, Finland). The mass measurementswere made using a precision balance (Shimadzu, model AW220,Japan). The incubations were done in a thermostated water bath(Marconi, model MA 184, Piracicaba, SP, Brazil). The centrifu-gations were performed in a Hitachi Koki centrifuge (modelCF15RXII, Hitachinaka, Japan) and absorbance readings weredone in a Shimadzu spectrophotometer (model UV1650TC,Japan) using the program UV Probe 2.21. The determinations ofhemoglobin (Hb), hematocrit (Ht), red blood cells (RBC), meancorpuscular volume (MCV), red cell distribution width (RDW),mean corpuscular hemoglobin (MCH), and mean corpuscularhemoglobin concentration (MCHC) were done with the use ofan automated system (Sysmex K4500, Sysmex Corporation,Mundelein, IL, USA). The determinations of total cholesterol

1378 | Anal. Methods, 2014, 6, 1377–1383

(t-C), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), VLDL-cholesterol (VLDL-C), triglycerides (TG) and glucose (Glu) weremade in an automatic analyzer (Hitachi 917, Roche Diagnostics,Indianapolis, IN, USA).

Standardization of the method to study the kinetics ofhemolysis

Initial tests for standardization of the method were performedin duplicated sets of microtubes (Eppendorf™) containing1 mL of 0.1, 0.3, 0.35, 0.4 or 0.5 g dL�1 NaCl. These solutionswere preincubated in a thermostated water bath at 37 �C for10 min. Aer addition of 20 mL of whole blood, the tubes werecapped, gently mixed and incubated at 37 �C. Lysis in each tubewas stopped by adding 1 mL of hypertonic solution of 5 g dL�1

NaCl aer 0–30 min. Aer incubating for 30 min under thesame conditions, the tubes were centrifuged for 10 min at1600 � g at 25 �C. This 30 min incubation was used only tomaintain the condition also present in the evaluation of theosmotic stability of erythrocytes.1,5,6,8,12,17,18 Aer centrifugation,the supernatants were carefully removed using an automaticpipette and subjected to absorbance readings at 540 nm. TheNaCl concentration of 0.4 g dL�1 was selected for the subse-quent tests, which were conducted under the same conditions,with interruption of lysis aer 0–30 min of incubation.

Determination of the kinetics of hemolysis

The kinetic parameters for the lysis of human erythrocytes weredetermined by statistical t to a hyperbola given by theequation:

A ¼ Amaxt

t1=2 þ t(1)

in which A is the absorbance at 540 nm obtained at each rangeof time (t), Amax is the maximum stationary value of absorbanceand represents the total lysis of erythrocytes, and t1/2 is the timeelapsed for the occurrence of half the total lysis (Amax/2).

Statistical analyses, calculations and data editing

The analyses of the hemolysis kinetics were made by applyingthe analytical routine of the integrated kinetic model ofMichaelis–Menten, using the soware GraphPad Prism 6(GraphPad Soware, San Diego, CA, USA), preset to automati-cally remove possible outlier points. All the other statisticalanalyses (normality test, Spearman correlations and ANOVA)were made using the program Origin 9.0 (Microcal Inc.,Northampton, Massachusetts, USA). The a priorimargin of error(E) estimated for this study (n ¼ 17), if we consider P ¼ 0.50, is23.8%, whichmeans that the sample data on which we base thisstudy can differentiate the population from which they wereextracted in a maximum of 23.8%.

Results

The results of initial tests to study the kinetics of lysis oferythrocytes, made in solutions of 0.1, 0.3, 0.35, 0.4 and 0.5 gdL�1 NaCl, with hypertonic interruption of haemolysis aer

This journal is © The Royal Society of Chemistry 2014

Table 2 Comparison of kinetic, haematological and biochemicalvariables (mean � standard deviation) between age groups

Variables 18–30 years (n ¼ 9) >40 years (n ¼ 8) p

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0–15 min is shown in Fig. 1. As the best resolution of the lysiscurve was obtained with 0.4 g dL�1 NaCl, the following testswere made in this salt concentration.

The descriptive statistics of the results obtained for thekinetic parameters (t1/2 and Amax), age, hemogram, and bloodlevels of glucose and lipids of the study participants are shownin Table 1. The search for normality by the Shapiro–Wilk testshowed that there was no normal distribution in the resultsobtained for t1/2, Amax, age, MCV, RDW, TG and VLDL-C(Table 1). However, aer stratication of the sample by agerange, the two subgroups obtained (18–30 and above 40 years)presented normal distribution for all the variables consideredin the study, according to the test of Shapiro–Wilk. A compar-ison between the age ranges with the use of ANOVA showed thatthe younger subgroup presented signicantly higher values oft1/2, RBC, RDW and HDL-C, while the older group presentedhigher values of MCV, MCH and Glu (Table 2).

Since the hemolysis kinetics has been properly tted by theintegrated kinetic model of Michaelis–Menten, it will be alsoproperly dened by the double reciprocal tting of Lineweaver–

Fig. 1 Kinetics of haemolysis (two determinations) obtained from theblood sample of a single volunteer (50 years old) at different NaClconcentrations (37 �C).

Table 1 Descriptive statistics of the study population

N Mean SD

t1/2 (min) 17 0.792 0.726Amax (DOD) 17 1.121 0.389Age (years) 17 40.412 21.552Hb (g dL�1) 17 14.171 2.017Ht (%) 17 43.059 5.987RBC (�106 mm�3) 17 4.865 0.657MCV (fL) 17 88.865 9.565RDW (%) 17 14.153 1.822MCH (pg) 17 29.259 3.430MCHC (g dL�1) 17 32.906 0.793Glu (mg dL�1) 17 88.553 15.884TG (mg dL�1) 17 100.471 36.135VLDL-C (mg dL�1) 17 20.201 7.223LDL-C (mg dL�1) 17 116.687 28.851HDL-C (mg dL�1) 17 49.859 13.724t-C (mg dL�1) 17 186.747 31.255

a At the 0.05 level, data were associated with a normal distribution (Shap

This journal is © The Royal Society of Chemistry 2014

Burk. Fig. 2 shows the linear regression lines for the two agesubgroups (18–30 and above 40 years) in a graph of 1/A540 nmversus 1/time. The experimental points of the two subpopula-tions were tted by statistically signicant and quite differentlines, certainly due to the signicant difference present betweenthe values of t1/2 of those groups (Table 2).

The association between all two-by-two combinations of thekinetic variables and the haematological and biochemicalvariables considered in this study was assessed by Spearmancorrelation (Table 3), since there was no normal distribution ofthe values of many of the variables considered in the study(Table 1). The values of t1/2 showed a signicant negativecorrelation with total cholesterol (t-C) and a negative borderlinecorrelation with LDL-C. In turn, the values of Amax showed

Median Minimum Maximum P

0.500 0.093 2.302 0.0071.218 0.264 1.502 0.00130 18 86 0.04214.5 9.6 17.8 0.983a

43.3 30.8 55.2 0.994a

4.83 3.89 6.27 0.590a

92.3 65.7 100.2 0.00413.2 12 18.9 0.03830.1 21 33.5 0.00833 31.2 34 0.414a

85 63 134 0.093a

94 57.2 183.1 0.01518.9 11.4 36.62 0.024103.6 78.2 177 0.119a

50.4 28.6 70.4 0.332a

182 130.9 233.2 0.315a

iro–Wilk test).

Age (years) 23.11 � 4.54 59.87 � 14.85 <0.0001a

t1/2 (min) 1.16 � 0.83 0.37 � 0.20 0.020a

Amax (DOD) 1.01 � 0.51 1.24 � 0.14 0.252Hb (g dL�1) 14.22 � 2.48 14.11 � 1.5 0.915Ht (%) 43.58 � 7.57 42.48 � 3.98 0.717RBC (�106 mm�3) 5.15 � 0.65 4.53 � 0.52 0.049a

MCV (fL) 84.61 � 11.1 93.65 � 4.34 0.047a

RDW (%) 15.33 � 1.77 12.83 � 0.41 0.001a

MCH (pg) 27.63 � 3.88 31.08 � 1.55 0.033a

MCHC (g dL�1) 32.64 � 0.82 33.2 � 0.70 0.155Glu (mg dL�1) 81.28 � 10.9 96.73 � 17.21 0.041a

TG (mg dL�1) 92.9 � 17.06 108.93 � 49.97 0.380VLDL-C (mg dL�1) 18.8 � 3.48 21.79 � 9.99 0.410LDL-C (mg dL�1) 105.47 � 23.12 129.30 � 30.8 0.089HDL-C (mg dL�1) 56.1 � 10.9 42.87 � 13.73 0.042a

t-C (mg dL�1) 180.36 � 28.5 193.92 � 34.56 0.389

a Statistically signicant difference (p < 0.05) between age groups (one-way ANOVA). At the 0.05 level, all variables were normally distributed(Shapiro–Wilk) in the age subgroups considered in study.

Anal. Methods, 2014, 6, 1377–1383 | 1379

Fig. 2 Double reciprocal fitting of the haemolysis kinetics of the twoage subgroups (18–30 years, n ¼ 9; 40–90 years old, n ¼ 8). Theexperimental points (mean � standard error of the mean) of the twosubpopulations were fitted by statistically significant lines. Conditions:0.4 g dL�1 NaCl and 37 �C.

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signicant positive correlations with the values of Hb, Ht, MCV,MCH and t-C, and a positive borderline correlation with thevalues of VLDL-C. Scatter plots of the signicant correlations oft1/2 and Amax with biochemical and haematological variables arepresented in Fig 3.

Discussion

The standardization of the kinetics of lysis of the erythrocytemembrane was developed from a series of preliminary tests withvarious modications during its standardization. These changesweremade tomake themethod a practical and viable tool for usein other studies. The development of the kinetic method wasbased on the principles used in the osmotic fragility test.

Initial tests for standardization of the method were carried outfrom a previous selection of four salt concentrations (0.3, 0.35, 0.4and 0.5 g dL�1 NaCl), near the intermediate region of a typicallysis curve obtained by incubation of erythrocytes under a xedtime condition, and another concentration (0.1 g dL�1 NaCl) quitefar from the lower salt concentration where complete lysis occursunder the same experimental conditions (Fig. 1). Although thisgure shows duplicate measurements of the spectral behavior ofthe blood sample collected from a single volunteer, in this phasethe testing of the inuence of salt concentration on the kinetics oflysis of human erythrocytes was done using blood samples from10 different volunteers. In all situations, similar proles, but withdifferent curves were obtained. The presentation of the results of asingle volunteer was a necessary choice, due to the heterogeneityof proles in the curves of different individuals. The variability inthe proles of these curves is a desirable feature and reects theability of themethod to respond to the wide range of factors whichcan affect the kinetics of hemolysis.

The concentrations of 0.3 and 0.35 g dL�1 NaCl did notproduce suitable lysis curves, because their plateaus (Amax) werereached very quickly. Similarly, the concentration of 0.50 g dL�1

1380 | Anal. Methods, 2014, 6, 1377–1383

NaCl was also not appropriate, because it did not enable theconstruction of a lysis curve under the conditions considered inthis study.

The curve obtained in 0.4 g dL�1 NaCl was the one whichshowed a more gradual and well-dened prole of lysis (Fig. 1) inorder tomake possible the determination of the kinetic variables.

Indeed, the kinetic variable t1/2 obtained in this way pre-sented signicantly different values between the two age groupsconsidered in this study (Table 2). The existence of this differ-ence means that the curves of lysis of the two age groupsshowed different patterns among themselves. Indeed, the exis-tence of differences in these patterns can be clearly seen in thedouble reciprocal graph of the haemolysis kinetics (Fig. 2).

Although this article concerns the standardization of amethod to study the kinetics of erythrocyte lysis, with thedetermination of the values of t1/2 and Amax, it is not yet a large-scale study to explore the broad spectrum of correlations thatthese parameters should have with a large group of variablesthat will include hematological and blood biochemical indicesand many other variables that will include not only cytoskeletalproteins and membrane phospholipids, but also the entirenetwork of blood and environmental factors that somehow hassome interaction or inuence on the erythrocyte.19

The haematological and biochemical parameters areimportant biomarkers to assist in the diagnosis of nutritionalstatus andmany diseases, as well as in assessing the response totreatment of these diseases.20 The use of the kinetics resultsobtained for the small population described in this study wasdone only to illustrate the potentiality of the method, ratherthan trying to nd all blood variables that can signicantlymodulate the hemolysis process. It is in this sense that thecorrelations presented in Table 3 should be regarded.

Amax values, which represent themaximum absorbance in thekinetics of hemolysis, showed the expected positive correlationswith Hb, Ht, MCH and MCV (Table 3). Amax also showed asignicant positive correlation with t-C and a borderline positivecorrelation with VLDL-C. Although these are expected correla-tions, the dispersion of points does not seem to unequivocallysupport their validities in some situations (Fig. 3).

The variable kinetic t1/2 was not correlated with the haema-tological variables, but showed a signicant negative correla-tion with t-C (Table 3 and Fig. 3). This must mean that elevatedcholesterol levels, within the limits of variation of bloodcholesterol in study participants, is contributing to acceleratethe kinetics of hemolysis, which would occur among olderindividuals, since t-C presented a positive correlation with age.

The volunteers within the age range of 40–90 years presentedlower erythrocyte (RBC) counts, conrming literature data thatshow a decrease in the amount of RBC with aging.20,21 Addi-tionally, the MCV values increased with age. MCV is the averagevolume of erythrocytes and is increased when there is nutri-tional deciency of folate and/or cobalamin, which is commonin older people.22 On the other hand, the increase in cell volumemay indicate a higher number of reticulocytes.23,24

Regarding the biochemical variables, higher values ofglucose in older people may be related to higher levels of gly-cation of proteins and a decrease in membrane uidity,25

This journal is © The Royal Society of Chemistry 2014

Tab

le3

Correlation(Spearman

)an

dsignifica

nce

matrixbetw

eenpairs

ofvariab

les

t 1/2

A max

Age

Hb

Ht

RBC

MCV

RDW

MCH

MCHC

Glu

TG

VLD

L-C

LDL-C

HDL-C

t-C

t 1/2

r p—

A max

r�0

.152

0p

0.56

04—

Age

r�0

.218

40.08

96p

0.40

000.73

24—

Hb

r�0

.261

20.76

52a

�0.162

1p

0.31

13<0

.001

0.53

43—

Ht

r�0

.284

30.72

30a

�0.258

90.98

96a

p0.26

870.00

100.31

57<0

.000

1—

RBC

r0.18

030.23

42�0

.530

4a0.67

42a

0.71

25a

p0.48

870.36

560.02

850.00

300.00

13—

MCV

r�0

.273

40.59

23a

0.39

470.32

210.26

36�0

.322

1p

0.28

820.01

220.11

690.20

740.30

660.20

74—

RDW

r0.26

26�0

.057

7�0

.617

3a0.09

640.14

230.47

27b

�0.259

7p

0.30

860.82

600.00

830.71

290.58

580.05

540.31

42—

MCH

r�0

.355

80.51

41a

0.41

59b

0.35

790.29

94�0

.299

00.93

86a

�0.365

5p

0.16

100.03

480.09

690.15

840.24

310.24

38<0

.000

10.14

915

—MCHC

r�0

.099

40.17

680.37

920.20

580.10

93�0

.056

50.20

95�0

.474

5b0.36

82p

0.70

410.49

730.13

330.42

820.67

630.82

940.41

980.05

430.14

59—

Glu

r�0

.206

00.08

830.68

94a

0.04

54�0

.019

6�0

.146

00.03

25�0

.415

0b0.11

910.29

55p

0.42

760.73

620.00

220.86

260.94

040.57

600.90

140.09

770.64

890.24

96—

TG

r�0

.174

00.40

440.15

710.52

12a

0.47

79b

0.39

98�0

.029

40.23

310.00

610.27

620.49

17a

p0.50

420.10

740.54

720.03

190.05

230.11

190.91

070.36

790.98

140.28

310.04

50—

VLD

L-C

r�0

.122

60.45

74b

0.17

740.55

46a

0.49

66a

0.39

260.04

170.21

800.07

920.35

690.46

13b

0.98

47a

p0.63

920.06

490.49

580.02

090.04

260.11

900.87

370.40

080.76

260.15

970.06

23<0

.000

1—

LDL-C

r�0

.453

4b0.31

370.38

040.41

080.39

220.14

100.01

10�0

.234

40.09

940.12

030.75

41a

0.59

31a

0.55

18a

p0.06

760.22

010.13

200.10

140.11

950.58

930.96

650.36

530.70

430.64

55<0

.001

0.01

210.02

17—

HDL-C

r�0

.014

70.34

80�0

.557

1a0.25

380.31

860.22

070.16

190.37

180.00

74�0

.593

0a�0

.584

9a�0

.232

8�0

.247

7�0

.183

8p

0.95

530.17

100.02

020.32

560.21

260.39

460.53

480.14

170.97

760.01

210.01

365

0.36

850.33

780.48

00—

t-C

r�0

.522

1a0.52

70a

0.25

640.52

73a

0.52

451a

0.11

280.24

89�0

.193

90.28

96�0

.033

20.52

48a

0.46

57b

0.44

64b

0.89

71a

0.15

93p

0.03

160.02

970.32

040.02

960.03

070.66

640.33

532

0.45

590.25

980.89

950.03

050.05

960.07

25<0

.000

10.54

14—

aStatistically

sign

icantcorrelations(p

<0.05

).bBorde

rlinecorrelations(0.05<p<0.10

).

This journal is © The Royal Society of Chemistry 2014 Anal. Methods, 2014, 6, 1377–1383 | 1381

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Fig. 3 Scatter diagrams observed in the correlations between kinetic and haematological variables of the study population (n ¼ 17).

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leading to changes in their structure and biological function.26

Thus, the mechanisms for maintaining the stability of themembrane would be affected, resulting in lower values of t1/2seen in the group above 30 years. In fact, De Arvelos andcolleagues found lower erythrocyte osmotic stability in indi-viduals with higher blood glucose.12

In general, older individuals have altered levels of plasmalipids, with high levels of LDL-C and low levels of HDL-C.20 Asthe composition of the erythrocyte membrane is inuenced byplasma levels of lipids,27 excess cholesterol can alter the physi-cochemical properties of the cell membrane.28,29 This agreeswith the signicant negative correlation obtained between t1/2and t-C and the lower levels of HDL-C observed in older indi-viduals of this study.

Conclusions

The study of the kinetics of hemolysis in 0.4 g dL�1 NaCl withinterruption of lysis with hypertonic solution of NaCl generateda gradual and suitable prole for obtaining the kinetic constantsof lysis Amax and t1/2. These constants presented sufficiently large

1382 | Anal. Methods, 2014, 6, 1377–1383

variances, among different individuals, in order to make themuseful in the study of modulating variables of the compositionand behavior of erythrocytes. The variable Amax, which repre-sents the largest concentration of hemoglobin of a volunteer,presented the expected direct correlations with haematologicalvariables Hb, Ht, MCH and MCV. The variable t1/2 was higher inthe subgroup of younger participants compared to the subgroupof older individuals. Furthermore, t1/2 showed a signicantnegative correlation with the blood levels of total cholesterol,which presented the already known positive correlation with theage of the participants. This must mean that the kinetic vari-ables obtained under the experimental conditions that werestandardized in this study are valid for the investigation offactors affecting the composition and behavior of erythrocytes.

Acknowledgements

We would like to thank FAPEMIG (CDS-APQ-01862-09, CDS-APQ-02025-10 and PPM-00485-12), CAPES (PE-PNPD AUX 2718/2011) and CNPq (307705/2012-9) for the nancial support thathas enabled this study.

This journal is © The Royal Society of Chemistry 2014

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