Tese de Mestrado Mestrado em Genética...

Tese de Mestrado Mestrado em Genética Molecular

Trabalho efetuado sob a orientação daDoutora Clara Sá MirandaE co-orientação daProfessora Doutora Maria João Marques Ferreira Sousa Moreira

iii

Apoio Financeiro

Este trabalho está inserido no projeto “Fabry Screen – Screening for Fabry disease

based on automatic classification of chromatographic images”, com a referência

PTDC/SAL‐BEB/100875/2008, financiado pela Fundação para a Ciência e a Tecnologia.

iv

Agradecimentos

Gostaria de começar por agradecer à Universidade do Minho pelos conhecimentos

transmitidos durante este ciclo de estudos e ao Instituto de Biologia Molecular e Celular, por

me acolher para realizar o projeto de mestrado. Um especial obrigado à Doutora Maria Clara

Sá Miranda e à Doutora Maria João Marques Ferreira Sousa Moreira pela orientação dos

trabalhos que constam nesta dissertação.

Pelo projeto em que estou inserida, e que em muito contribuiu para esta dissertação,

fico grata à Unidade do Lisossoma e do Peroxissoma, liderada pela Doutora Maria Clara Sá

Miranda, e ao “Bioimaging group”, liderado pela Doutora Ana Mendonça.

Ao Daniel devo os ensinamentos sobre TLC e a energia com que sempre se prontificou

a ajudar-me na resolução de problemas e na interpretação das minhas placas.

Pela paciência e pelos conhecimentos transmitidos, agradeço ao Rui, à Lorena, à

Andrea, ao Paulo e à Fátima.

Ainda ao Paulo e ao Rui, bem como à Cátia, à Cláudia, à Marta, à Tânia e à Ana,

agradeço o ombro amigo, os conselhos, e os momentos da mais pura diversão que permitiam

superar qualquer frustração.

Um enorme obrigado aos meus pais, pelo incentivo, força e amor.

v

HPTLC Detection of Gb3 in Urine Samples: improvement of the method

and sampling

Abstract

Fabry disease (FD) is a lysosomal storage disorder caused by deficient or null

production of the enzyme α-galactosidase A (α-Gal A) as a result of mutations in α-

galactosidase A gene (GLA), located on the X chromosome. The systemic accumulation of α-

Gal A substrates, which are excreted in urine, leads to progressive damage of several organs

and tissues and ultimately results in death due to heart failure, renal failure and

cerebrovascular accidents.

Many studies are focused in diagnosing FD through measurement of α-Gal A activity in

blood and of accumulated substrates, mainly globotriaosylceramide, in plasma or urine. Mass

spectrometry techniques are extensively used for screening of FD and other lysosomal storage

disorders. However, mass spectrometry is an expensive method, requires a specialized

operator and involves complex equipment. Thin-layer chromatography, on the other hand, is a

robust and well explored method suitable for analysis of a large number of samples.

The collection of urine samples is easy and non-invasive, which facilitates the

cooperation of the subjects, but urine samples are difficult to transport without degradation.

In this study, the problematic of urine samples transport was overcome by using filter

paper as a supporting material that allows shipping through general mail without deterioration.

The high-performance thin-layer chromatography method conventionally used to analyse Gb3

in urine samples was successfully optimised to analyse urine in filter paper. Using this method

60% of the previously diagnosed FD patients revealed increased Gb3 levels and one patient on

enzyme replacement therapy showed significant Gb3 reduction after three months of therapy.

The developed screening method offers an easy and simple method for urine sample

shipping without degradation and suitable for a mass screening and ERT follow up program.

vi

HPTLC Detection of Gb3 in Urine Samples: improvement of the method

and sampling

Resumo

A doença de Fabry (FD) é uma doença lisossomal de sobrecarga causada pela

atividade deficiente da enzima α-galactosidase A (α-Gal A) do, como resultado de mutações no

gene desta enzima (gene GLA), localizado no cromossoma X. A acumulação sistémica dos

substratos da α-Gal A, que são excretados na urina, leva à lesão progressiva de vários órgãos e

tecidos, conduzindo por fim à morte devido a falência cardíaca, falência renal e acidentes

vasculares cerebrais.

Vários estudos estão debruçados no diagnóstico da doença de Fabry através da

medição da atividade da α-Gal A em sangue e de substratos acumulados, principalmente o

globotriaosilceramida, em urina e plasma. As técnicas de espectrometria de massa são

extensivamente no rastreio da doença de Fabry e outras doenças lisossomais de sobrecarga.

Contudo, a espectrometria de massa é um método dispendioso, requer um operador

especializado e envolve equipamentos complexos. A cromatografia em camada fina, por sua

vez, é um método robusto e bem explorado adequado à análise de grandes grupos de

amostras.

A colheita de amostras de urina é fácil e não é invasiva, o que facilita a cooperação

por parte dos dadores, mas as amostras de urina são difíceis de transportar sem que haja

deterioração.

Neste estudo, a problemática do transporte de amostras de urina foi ultrapassada

utilizando o papel de filtro como material de suporte, permitindo que as amostras sejam

enviadas por correio normal sem degradação. O método de cromatografia em camada fina de

alta performance convencionalmente utilizado na análise de Gb3, em amostras de urina, foi

otimizado com sucesso para analisar amostras de urina em papel de filtro. Recorrendo a esta

metodologia, verificou-se que os níveis de Gb3 estavam aumentados em 60% dos doentes de

Fabry previamente diagnosticados, e que um paciente em terapia enzimática de substituição

mostrou uma redução significativa de Gb3 após três meses de terapia.

O método de rastreio desenvolvido oferece um modo simples e fácil de envio das

amostras de urina sem que haja degradação, sendo por isso, adequado para o rastreio em

massa e seguimento dos pacientes em terapia.

vii

INDEX

ABBREVIATION LIST IX

ILLUSTRATION LIST XI

TABLE LIST XII

1. INTRODUCTION 1

1.1. An Overview 2

1.2. Molecular Genetics 3

1.3. Metabolic Background of Fabry Disease 5

1.4. Glycosphingolipid Storage in a Cellular and Subcellular Perspective 9

1.5. Clinical Profile and Pathophysiology 11

1.5.1. Heterozygotes 14

1.6. Treatment 16

1.7. Laboratory Diagnosis of Fabry Disease 17

2. STUDY AIMS 20

3. SAMPLES AND METHODOLOGY 22

3.1. Sampling 23

3.1.1. Biological samples 23

3.1.2. Sample Collection 24

3.2. Reagents and Standards 25

3.3. Methods 26

3.3.1. High-Performance Thin-Layer Chromatography for Conventional Urine Samples 26

4. RESULTS 28

viii

4.1. High-Performance Thin-Layer Chromatography Optimization for Filter Paper Urine Samples 29

4.1.1. Validation of Filter Paper for HPTLC Analysis 29

4.1.2. Extraction Method 31

4.1.3. Lipid Fractionation Process 35

4.1.4. Development of the HPTLC Plate 36

4.1.5. Spraying Solution 37

4.1.6. Gb3 Detection Limit 39

4.1.7. Lipid loss in the HPTLC technique 40

4.1.8. Optimised Protocol 41

4.2. Lipidic Profile of FD Patients and Control subjects 43

4.3. Effect of ERT in FD Patients 46

5. DISCUSSION 48

6. CONCLUSIONS AND FUTURE PERSPECTIVES 53

7. REFERENCES 55

8. APPENDIX I 71

9. APPENDIX II 75

ix

Abbreviation list

3-KSR 3-ketosphinganine reductase

α-Gal A (GLA) α-galactosidase A

β-Hex A (B) β-Hexosaminidase A (B)

AC acid ceramidase

ASA arylsulfatase A

aSMase acid sphingomyelinase

C/M chloroform/methanol

Cer 4 asialoganglioside GM1

CerS ceramide synthase

CERT ceramide transfer protein

CGT ceramide UDP-galactosyltransferase

CST galactose-3-osulfotransferase

CTH ceramide trihexoside

DBS dried blood spot

DHCer dihydroceramide

DHCerS dihydroceramide desaturase

ELISA enzyme-linked immunosorbent assay

ER endoplasmic reticulum

ERT enzyme replacement therapy

FAPP2 four-phosphate adaptor protein 2

FD Fabry disease

FOS Fabry outcome survey

GALC GalCer-β-galactosidase

GalCer galactosylceramide

Gb3 globotriaosylceramide

Gb3 synthase α-1,4-galactosyltransferase

Gb4 globotetraosylceramide; globoside

GCase β-glucocerebrosidase

GCS glucosylceramide synthase

x

GLA α-galactosidase A gene

GLC gas-liquid chromatography

GlcCer glucosylceramide

GM1-GLB GM1-β-galactosidase

GM3 synthase lactosylceramide α-2,3-sialyltransferase

GSL glycosphingolipids

GT3 synthase α-N-acetylneuraminide α-2,8-

sialyltransferase

HPLC high-performance liquid chromatography

HPTLC high-performance thin-layer

chromatography

LacCer lactosylceramide

LacCer synthase LacCer synthase

LVH left ventricular hypertrophy

Lyso-Gb3 globotriaosylsphingosine

Lyso-GSLs lyso-glycosphingolipids

MS mass spectrometry

nSMase neutral sphingomyelinase

PE phosphatidylethanolamine

RSD relative standard deviation

S1P sphingosine 1-phosphate

S1-P Pase sphingosine 1-phosphate phosphatase

(Sa) N-ACT sphinganine N-acyltransferase

Sa1P sphinganine 1-phosphate

Sap A (B, C, D) saposin A (B, C, D)

SD standard deviation

Sk sphingosine kinase

SMS sphingomyelin synthase

SM sphingomyelin

SPT serine palmitoyl transferase

SRT substrate reduction therapy

TLC thin-layer chromatography

xi

Illustration list

FIGURE 1: THE GSL METABOLIC PATHWAYS ........................................................................................................... 6

FIGURE 2: STRUCTURES OF GLOBOTRIAOSYLCERAMIDE AND LYSO-GLOBOTRIAOSYLCERAMIDE ............................................ 7

FIGURE 3: SCHEMATIC REPRESENTATION AND LOCALIZATION OF GSL METABOLISM IN THE CELL ....................................... 8

FIGURE 4: LIPID AGGREGATES IN FABRY DISEASE ..................................................................................................... 9

FIGURE 5: SOME CELL TYPES KNOWN TO EXPRESS HIGH DEGREE OF STORAGE IN FABRY DISEASE ....................................... 10

FIGURE 6: SCHEMATIC MODEL FABRY DISEASE PROGRESSION .................................................................................... 11

FIGURE 7: REPORTED SIGNS AND SYMPTOMS OF FABRY DISEASE AMONG 366 FD PATIENTS ............................................. 15

FIGURE 8: CONVENTIONAL COLLECTION URINE FLASK .............................................................................................. 25

FIGURE 9: FILTER PAPER KIT ............................................................................................................................... 25

FIGURE 10: FILTER PAPER KIT INSTRUCTION SHEET FOR COLLECTION OF URINE SAMPLES ................................................ 73

FIGURE 11: ANALYSIS OF 1 AND 2 FILTER PAPERS FROM A CONTROL SAMPLE................................................................ 30

FIGURE 12: FD PATIENTS’ ANALYSIS OF CONVENTIONAL 20 ML URINE SAMPLES AND URINE SAMPLES

APPLIED TO FILTER PAPERS ............................................................................................................... 31

FIGURE 13:EVALUATION OF DIFFERENT EXTRACTION CONDITIONS ............................................................................... 32

FIGURE 14:ANALYSIS OF DIFFERENT EXTRACTION AGITATION METHODS AND ORGANIC SOLVENT SOLUTIONS ......................... 34

FIGURE 15:ELUTION OF NEUTRAL LIPIDS FRACTION ................................................................................................. 35

FIGURE 16:DEVELOPMENT OF THE HPTLC PLATE FOR 7 CM ..................................................................................... 37

FIGURE 17:VISIBLE WHITE BANDS IN PATIENTS AND CONTROL URINES ......................................................................... 37

FIGURE 18: ORCINOL STAINING OF NEUTRAL LIPIDS ................................................................................................. 38

FIGURE 19: ESTIMATION OF THE GB3 DETECTION LIMIT ........................................................................................... 39

FIGURE 20:GB3 LOSS DURING THE HPTLC TECHNIQUE AND FRACTIONATION OF THE OPTIMISED AND

CONVENTIONAL METHOD .................................................................................................................. 40

FIGURE 21: REPRODUCIBILITY EVALUATION OF THE OPTIMISED METHOD....................................................................... 42

FIGURE 22: ANALYSIS OF SAMPLES FROM CONTROL SUBJECTS AND FD PATIENTS BY THE OPTIMISED

HPTLC METHOD ............................................................................................................................ 44

FIGURE 23:ANALYSIS OF SAMPLES FROM FD PATIENTS COLLECTED BEFORE AND DURING ERT ......................................... 47

xii

Table list

TABLE I: EARLY SIGNS AND SYMPTOMS OF FABRY DISEASE ........................................................................................ 12

TABLE II: COHORT CHARACTERISTICS ................................................................................................................... 23

TABLE III: CHARACTERISTICS OF CONTROL SUBJECTS ............................................................................................... 72

TABLE IV: CHARACTERISTICS OF FD PATIENTS ........................................................................................................ 24

TABLE V: CONSTITUTION OF A STANDARD MIXTURE FOR HPTLC ................................................................................. 27

TABLE VI: DENSITOMETRY OF GB3 AND SM+GB4 BANDS FROM THE FILTER PAPER SAMPLES ........................................... 30

TABLE VII: DENSITOMETRY OF GB3 AND SM+GB4 BANDS FROM URINE SAMPLES EXTRACTED UNDER

DIFFERENT CONDITIONS ........................................................................................................................ 32

TABLE VIII: DENSITOMETRY OF GB3 BANDS FROM EMPTY FILTER PAPERS SUBJECTED TO DIFFERENT

EXTRACTION AGITATION PROCEDURES ...................................................................................................... 76

TABLE IX: DENSITOMETRY OF GB3 ELUTED WITH 8 ML OR 16 ML OF ORGANIC SOLVENT SOLUTION DURING

FRACTIONATION................................................................................................................................... 36

TABLE X: DENSITOMETRY FOR CALCULATION OF GB3 LOSS DURING THE HPTLC TECHNIQUE AND

FRACTIONATION WITH THE OPTIMISED AND CONVENTIONAL METHODS ............................................................. 41

TABLE XI: ASSESSMENT OF VARIABILITY OF THE OPTIMISED METHOD BY DENSITOMETRY................................................... 43

TABLE XII: DENSITOMETRY OF CONTROL AND FD PATIENTS WITH TRACEABLE AMOUNTS OF GB3 ....................................... 45

TABLE XIII: DENSITOMETRY OF GB3 AND SM BANDS OF FD PATIENTS AND CONTROL SUBJECTS ....................................... 76

CHAPTER I

1. INTRODUCTION

HPTLC Detection of Gb3 in Urine Samples: improvement of the method and sampling

Chapter I - Introduction

[2]

1.1. An Overview

Fabry disease (OMIM #301500) is an X-linked inherited lysosomal storage disorder

caused by α-galactosidase A (EC 3.2.1.22) deficiency. α-Gal A is responsible for cleaving out

neutral glycosphingolipids with α-galactosyl residues and is codified by the GLA gene (OMIM

#300644), located on Xq22.1 (Desnick et al, 2001). The impairment of its activity results in

progressive visceral accumulation of its substrates, mainly globotriaosylceramide (Gb3 or

CTH), digalactosylceramide, and to a lesser extent, globotriaosylsphingosine (lyso-Gb3), blood

group B, B1 and P1 related glycolipids (Hrebícek & Ledvinová, 2010). This accumulation in

lysosomes leads to dysfunction of basic metabolic processes in the cell, leading to its death,

which contributes to the development of secondary (inflammatory) processes culminating with

vital organ dysfunction (Eng et al, 2007). Gb3 is the most abundant accumulated substance

making it an appropriate molecular marker of FD. Increased values of Gb3 were reported in

both hemizygous males and heterozygous females (Bodamer, 2007). Even though FD follows

an X-linked pattern of inheritance, having males more severe symptoms, heterozygous females

that in the past were considered as carriers, can be as affected as their male counterparts.

About 69,4% of the 1077 females in the Fabry Registry (an ongoing observational database

that tracks natural history and outcomes of FD patients) had symptoms and signs of the

disease, showing that females have a considerable risk for major organ involvement and

decreased quality of life (Germain et al, 2010).

FD is estimated to have a prevalence ranging from 1:40000 males to 1:117000 births

(Germain, 2010; Levy & Feingold, 2000; Meikle et al, 1999). Due to its rarity, accurate values

about its prevalence are harder to obtain. FD was for a long time predominantly described in

males developing a “classic” phenotype (compatible with null α-Gal A activity and multi-organ

involvement). However the remarkable heterogeneity in clinical manifestations among the

patients, even within the same pedigree, led to the creation of sub-classifications, such as, the

“cardiac variant” and the “renal variant”, for those patients showing predominant or exclusive

cardiac or renal complications (Germain, 2010; Hrebícek & Ledvinová, 2010).

Despite the fact that the first signs of the disease emerge during childhood,

glycosphingolipid accumulation processes might start in the fetal stage. The clinical features

observed in FD patients worsen in adulthood and the mortality causes are generally related to



[3]

heart failure, renal failure or cerebrovascular accidents (Liu et al). Furthermore, FD signs and

symptoms are not pathognomonic, which can delay the clinical diagnosis often until irreversible

organ damage is already present. This becomes more evident in the absence of family history

and in the case of affected females, since the range of enzyme activity in these patients is

broader than in males (Germain, 2010). While FD can be diagnosed in males by demonstrating

significantly reduced enzymatic activity of α-Gal A in plasma, leukocytes, fibroblasts or dried

blood spots (DBSs; Gaspar et al, 2010), heterozygous females can only be unequivocally

identified by mutational analysis. Classic hemizygotes and over 90% of the heterozygous

females show elevated urinary Gb3 levels (Winchester & Young, 2006). For male patients with

residual α-Gal A activity or with an atypical clinical presentation, measurement of urinary Gb3

and sequencing of the whole GLA gene must be carried out (Winchester & Young, 2006).

Although there is still no cure for FD, enzyme replacement therapy (ERT) is available in

Europe since 2001. Two different products are approved, Agalsidase alfa (Replagal®, Shire

Human Genetic Therapies) and Agalsidase beta (Fabrazyme®, Genzyme Corporation; Lidove et

al, 2010).

1.2. Molecular Genetics

The GLA gene is localized at the X chromosome in the region Xq22.1 and is

constituted by 12 kilobases distributed through 7 exons (Bishop et al, 1986; Kornreich et al,

1990).

Above 400 point mutations and 150 small rearrangements have been identified

scattered throughout the GLA gene (Gal, 2010; Masson et al, 2004). Most of the identified

patients have different mutations. The great majority of the GLA mutations are private/unique

of the family (Gal et al, 2006; Schäfer et al, 2005).

The point mutations include missense, nonsense and splice site mutations, and the

rearrangements include deletions and intragenic insertions (Desnick et al, 2001; Gal, 2010).

The missense and nonsense mutations correspond to approximately 70% of the described

mutations known to cause FD (Schäfer et al, 2005). The high proportion of novel mutations,

the large phenotypic heterogeneity associated with the same mutation and the fact that

patients with FD may develop disease-related complications observed in the general



[4]

population, difficult the genotype-phenotype correlations (Ries & Gal, 2006). However, some

phenotypes associated to Fabry disease may be attributed to specific mutations of the GLA

gene (Brown et al, 1997; Eng et al, 1993; Germain et al, 2002; Saito et al, 2011; Spada et al,

2006). Alleles with nonsense and splice-site mutations in the GLA gene or out-of-frame short

rearrangements usually lead to a premature termination codon. Consequently, these alleles do

not produce the enzyme. The missense mutations can affect the enzyme activity in a quite

variable way. Clearly, the substitution of one residue in the pocket of the enzyme, or

substitution of a residue essential for proper three-dimensional folding of the protein, generally

results in a completely inactive enzyme. In contrast, the missense mutations associated with a

less severe phenotype are generally located away from the active site of the enzyme, resulting

in small structural changes. However, the enzyme product of missense mutations may still be

subject to post-translational inactivation and degradation (Gal, 2010; Matsuzawa et al, 2005).

In Fabry disease, heterozygous females where initially referred to as carriers

concerning their genotype. Even so, the affected females can present a phenotype that ranges

from asymptomatic to the classical form of the disease seen in the majority of the male

patients (MacDermot et al, 2001b; Wang et al, 2007; Whybra et al, 2001; Wilcox et al, 2008).

Among females presenting the classic form of FD (null α-Gal A activity), missense and

nonsense mutations are present. On the other hand, in patients with less severe or

asymptomatic forms of the disease, only missense mutations are present, which leads to

residual activity of α-Gal A (Desnick et al, 2001). Nevertheless, it is harder to predict the

hetezygotes phenotype due to the X-inactivation process, which occurs early in the female

embryonic phase and leads to a random and almost complete inactivation of one of the X

chromosomes per cell. Therefore, depending on which α-Gal A gene is inactivated (the normal

or the mutated gene) and according to the skewing rate that occurred at the precedent cell,

different organs from the same patient may have distinct pathology degrees (Elstein et al,

2012).

Since FD is linked to the X chromosome, males descending from hemizygous males do

not carry the mutated gene, while females acquire the mutated gene. Therefore, prenatal

diagnosis is of major importance. FD can be determined by assessing α-Gal A activity in

amniotic fluid at weeks 12 to 14 (Desnick et al, 2001).



[5]

1.3. Metabolic Background of Fabry Disease

The deficient α-Gal A catalytic activity of Gb3 and other substrates, leads to subcellular

functional alterations responsible for the massive lysosomal storage of these components.

Ultimately, the storage culminates in irreversible cell damage and death (Elleder, 2010).

α-Gal A substrates are sphingolipids characterized by an α-galactosyl moiety, of which

Gb3, a glycolipid of the globo-series metabolism, is the major accumulated substrate. The

massive storage of this GSL is pointed out as the major responsible for FD development. As

seen in Figure 1, Gb3 can be synthesized from lactosylceramide (LacCer) by Gb3 synthase or it

can be a product of globotetraosylceramide (Gb4) degradation by β-Hexosaminidase A (B) (Xu

et al, 2010).

Besides Gb3, also di-galactosylceramide possesses an α-galactosyl moiety, making it

suitable for degradation by α-Gal A. This glycolipid and other glycolipids from the blood group

B, like the B1 and P1 related lipids, with α-galactosyl extremities have been shown to

accumulate in Fabry patients, although in much lesser extent. Patients with this biochemical

profile do not seem to be in higher risk for developing severe disease manifestations. Therefore

scientific attention is mostly turned to the study of the glycosphingolipids that are thought to be

related to the disease progression and severity (Elleder, 2010).

Lyso-Gb3, a deacylated derivate of Gb3 is also accumulated in FD (Aerts et al, 2008).

Lyso-Gb3 has a similar structure to that of Gb3 but instead of ceramide it has a sphingosine

backbone (Figure 2).

HPTLC Detection of Gb3 in Urine Samples: improvement of the method and sampling Chapter I - Introduction

[6]

Figure 1: The GSL metabolic pathways. The synthesis and degradation of GSLs are sequential processes. The synthesis is catalyzed by membranous glycosyltransferases in the endoplasmic reticulum (ER). GSL synthesis

begins with condensation of serine and palmitoyl-CoA, leading to de novo biosynthesis of ceramide. Ceramide can also be formed from sphingosine by addition a fatty acid chain of specific length, or hydrolysis of sphingomyelin

(SM). Once formed, ceramide can originate galactosylceramide (GalCer) or glucosylceramide (GlcCer) by addition of galactose or glucose residues, respectively. GalCer can form sulfatide and di-galactosylceramide (one of the

GSLs accumulated in Fabry patients). The addition of one β-gal residue to GlcCer originates LacCer, which is a precursor of Gb3. Gb3 is created when a α-gal is added to LacCer and Gb4 is formed when α-gal is added to Gb3.

1=GM2/GD2/GA2/GT2-synthase. The degradation reactions occur in the lysosome by action of several hydrolases. (Adapted from Xu et al, 2010).

Synthesis

Degradation


Chapter II – Study Aims

[7]

Figure 2: Structures of globotriaosylceramide and lyso-globotriaosylceramide. A. Gb3 is organized in 3 sugar

residues, α-gal, β-gal and β-glc, and a ceramide tail composed of a fatty acid acyl chain, that varies in length and saturation,

and a sphingosine backbone. B. In lyso-Gb3, a deacylated derivate of Gb3, the fatty acid chain is missing (Adapted from

Hřebíček & Ledvinová, 2010 and Cole et al, 2007).

The deposition of other lyso-glycosphingolipids (lyso-GSLs) was shown to be a

characteristic aspect of LSDs, such as Krabbe (deposition of galactosylsphingosine) and

Gaucher disease (deposition of glucosylsphingosine; Nilsson & Svennerholm, 1982; Orvisky et

al, 2002; Svennerholm et al, 1980). These lyso-GSLs are described for their toxic effects and

as potential pathologic agents in these diseases (Igisu & Suzuki, 1984; Orvisky et al, 2002).

Therefore, lyso-Gb3 could be implicated in FD pathological events as well. In 2008, Aerts and

colleagues described high levels of lyso-Gb3 in plasma of Fabry patients and mice. They

advanced that this compound competes with Gb3 for the α-Gal A activity and promotes smooth

muscle cell proliferation, which can be related to an increase in thickness of the intima-media

of the heart (Aerts et al, 2008).

The understanding and knowledge of the pathways related to the incorporation and

degradation of these GSLs may lead to relevant discoveries in the mechanisms associated to

disease progression and tissue/cell specific reaction processes. The so far described synthesis

and degradation pathways can be seen in Figure 3. The pathways that lead to α-Gal A



[8]

substrate delivery into the lysosome for degradation are still poorly understood but it is known

that the substrates can be localized at the cell membrane or at the extracellular matrix and

then be transported by endocytosis by plasma lipoproteins (Elleder, 2010; Huwiler et al,

2000). Thereafter, the endocytic vesicles are fused with the lysosome where α-Gal A plays its

role, generating LacCer as a product of GSL degradation. The internalization of α-Gal A

substrates into the cell is also possible through phagocytosis of old or damaged red blood cells

(Elleder, 2010). Therefore, understanding and exploring the possible mechanisms of GSL

trafficking may be important to predict the reaction of tissues and cells in their current

environment and in response to several conditions.

Figure 3: Schematic representation and localization of GSL metabolism in the cell. GSL biosynthesis starts in the ER. Ceramide

can be synthesized de novo from L-serine condensation with palmitoylCoA on the cytoplasmic face of ER or it can be formed from the GSLs

recycled in the lysosome, specifically sphingosine. After this processes, ceramide can follow three possible pathways: a) it can form GalCer in

the ER which on the other hand will generate sulfatide in the Golgi complex; b) it can be transported to the golgi complex in vesicles where it

originates GlcCer on its cytoplasmic face; or c) ceramide can be transported to the mid-Golgi by CERT (ceramide transfer protein) to SM

synthesis in the Golgi lumen. The FAPP2 (four-phosphate adaptor protein 2) enables the transport of GlcCer between ER and Golgi complex.

GlcCer also reaches the cell membrane but the inherent mechanisms from which this happens are still unknown. FAPP2 is capable of

transferring GlcCer from the cis face to the trans face of the Golgi complex as well, allowing the formation of a large variety of GSLs.

Synthesized GSLs are then exocytosed from the cell to the extracellular matrix. Membrane GSLs recycling process starts by endocytosis and

fusion with a lysosome for their degradation to sphingosine and free fatty acids. Membrane and cytosolic hydrolysis of SM can generate

sphingosine as well which is further degraded. (Adapted from Xu et al, 2010).



[9]

1.4. Glycosphingolipid Storage in a Cellular and Subcellular

Perspective

α-Gal A deficiency is correlated with appearance of lipid deposits inside the cells, more

specifically within lysosomes. When GSLs start to accumulate, lysosomes acquire a

conformation characterized by stacks of membranous structures with concentric or parallel

disposition (zebra bodies; Figure 4). The circular deposits and fused lysosomes can be

observed by convergence of two or more distinct laminar aggregate sets (Figure 4). Although

the lipid aggregates entirely occupy the lysosome lumen and force lysosome expansion, no

alterations are currently described for the biology of the lysosome. In 2008 Valbuena and

coworkers hypothesized that the rupture of the storage lysosomes into the cytoplasm may be

sufficient to cause cell death (Valbuena et al, 2008).

Figure 4: Lipid aggregates in Fabry disease. Photomicrograph of a kidney biopsy representing GSL deposits: in cells of

distal tubules (T) and in a capillary (C). Fused lysosomes (FL). (Germain, 2010).

The cell types affected by GSL storage in Fabry disease include endothelial cells,

smooth muscle cells, cardiomyocytes, peripheral neurons and perineural cells, fibroblasts,

pericapillary cells (i.e. pericytes and podocytes), ciliary epithelial cells, renal tubular epithelial

cells, some secretory cells, macrophages and adipocytes (Figure 4 and 5, Chimenti et al,

FL

FL

FL



[10]

2008; Elleder, 2003; Elleder, 2010; Hůlková & Elleder, 2010). Leukocytes are also affected in

FD showing Gb3 deposits and physiologic alterations (Macedo et al, 2012; Rozenfeld et al,

2009a)

Figure 5: Some cell types known to express high degree of storage in Fabry disease. (a) complex capillary

endothelium-pericyte; (b) glomerular podocyte; (c) smooth muscle cell; (d) subcutaneous adipocytes (asterisks); (e) peripheral

neuron in the bronchial wall; perineurial cells in large (f) and terminal (g) nerve fibres (magn. a, b, c, g 4,000×, f 3,000×).

SC – collapsed storing capillary. (Adapted from Elleder, 2010 and Hůlková & Elleder, 2010).

d



[11]

The extension of cell damage and the number of affected cells within an organ will

determine the burden of the disease. These factors worsen as the disease progresses resulting

in dysfunction of several organs (Figure 6; Eng et al, 2007).

Figure 6: Schematic model of Fabry disease progression. (Eng et al, 2007).

1.5. Clinical Profile and Pathophysiology

The pathological processes and the majority of the clinical manifestations become

more severe with aging, however some of the pathological events have an early onset and can

be detected at a pediatric age (see Table I). FD has a wide spectrum of clinical manifestations

and systemic organ commitment is observed in patients with the classic phenotype (patients

with null α-Gal A activity). The clinical profile of these patients involves left ventricle hypertrophy

(LVH), acute and chronic neuropathic pain, renal insufficiency, skin lesions, ocular alterations

and gastrointestinal symptoms (Germain, 2010).

The renal insufficiency develops as a consequence of cellular lesions caused by Gb3

storage, mainly in the glomerulus and podocytes, but damage also occurs in glomerular

endothelial, mesangial and parietal epithelial cells (Alroy et al, 2002). This background evolves

to mesangial widening and, in later stages, to glomerulosclerosis. Alterations in distal tubular

epithelial cells and, in less extent, in proximal tubular epithelial cells, in addition to vascular

lesions, will progressively cause tubular atrophy and interstitial fibrosis (Alroy et al, 2002).



[12]

Proteinuria results as a sign of renal function impairment and in adulthood renal insufficiency

occurs due to chronic kidney disease, leading the patient to hemodyalisis (Desnick et al,

2003).

Table I: Early signs and symptoms of Fabry disease. (adapted from Eng et al, 2007).

A renal variant of FD was detected in a Japanese study, where hemodialysis patients

with glomerulonephritis presented absent or low α-Gal A activity. One novel and two previously

described mutations in the GLA gene were identified (Nakao et al, 2003). In this variant, some

FD patients present disease manifestations confined to the kidney, but they may also develop

vascular disease of the heart or brain later in life (Germain, 2010).

FD also promotes the development of cardiomyopathies, like LVH, arrhythmias,

conduction and valvular abnormalities, and coronary heart disease (Germain, 2010). The

Organ system Sign/Symptom

Nervous system Acroparesthesias

Nerve deafness

Heat intolerance

Hearing loss, tinnitus

Gastrointestinal tract

Nausea, vomiting, diarrhea

Postprandial bloating and pain, early satiety

Difficulty in gaining weight

Skin Angiokeratomas

Hypohidrosis

Eyes Corneal and lenticular opacities

Vascolopathy (retina, conjuntiva)

Kidneys Microalbuminuria, proteinuria

Impaired concentration ability

Hyperfiltration

Increased urinary Gb3 excretion

Heart Impaired heart rate variability

Arrhythmias

Electrocardiogram abnormalities

Mild valvular insufficiency



[13]

cellular processes leading to these alterations develop throughout patient’s life, with

accumulation of GSLs, particularly Gb3, on coronary arteries, myocardium, endocardium, and

intravascular and intracardiac nerves (Askari et al, 2007).

Myocardial fibrosis is another pathological process described in FD patients (Hasegawa

et al, 2006; Moon et al, 2006). In advanced cases, transmural replacement fibrosis evolves to

a congestive heart failure (Germain, 2010).

The heart is predisposed to the GSL storage and in some cases it can be the only

affected organ. This particular phenotype of FD is described as the cardiac variant of the

disease and it is the most frequent atypical variant. Manifestations include cardiomegaly,

electrocardiographic abnormalities consistent with cardiomyopathy, non-obstructive

hypertrophic cardiomyopathy and myocardial infarctions (Germain, 2010).

Vascular lesions caused by GSL excessive storage origin angiokeratomas, which are

characterized by single or clustered small reddish purple, raised superficial skin lesions.

Generally this manifestation can be localized on the buttocks, groin, umbilicus and upper

thighs (Germain, 2010). Skin sweat glands are affected by FD as well, leading to anhydrosis or

hypohydrosis, which can cause heat and exercise intolerance (Eng et al, 2006; Kang et al,

1987; Orteu et al, 2007; Shelley et al, 1995).

Cornea opacities, commonly called cornea verticillata, and a corneal haze, are present

in many FD patients but do not affect the vision capacity (Orssaud et al, 2003). Conjunctival

and retinal vascular tortuosity can be observed in some patients as well as anterior and

posterior subcapsular cataracts (Nguyen et al, 2005; Orssaud et al, 2003; Sodi et al, 2007).

The posterior subcapsular cataract is considered the only pathognomonic clinical event of FD,

therefore described as “Fabry cataract” (Germain, 2010). Optic neuropathy was also detected

in FD patients in a pilot study (Pitz et al, 2009).

Cerebrovascular complications strongly contribute to morbidity and mortality of FD

patients and are related to lesions in the large and small vessels, and alterations in other

elements of the Virchow’s triad (which include factors that are thought to contribute to

thrombosis, being the endothelial injury of brain vessels one of them; Fellgiebel et al, 2006;

Moore et al, 2007). The most preoccupying manifestations observed in FD patients are

transient ischemic attacks and strokes, that include ischemic and hemorrhagic stroke, and

both have onset around the forth decade of life (Schiffmann et al, 2009). Symptoms like



[14]

headache, vertigo/dizziness and vascular dementia have also been reported (Mendez et al,

1997; Mitsias & Levine, 1996). Besides the clinical manifestations FD patients also develop

periventricular white-matter lesions, microbleeds, cortical grey-matter infarcts and deep lacunar

infarcts in both grey and white matter (Buechner et al, 2008; Jardim et al, 2004; Sims et al,

2009).

Gastrointestinal symptoms are common between FD patients and include post-prandial

abdominal pain, diarrhea, nausea and vomiting (MacDermot et al, 2001a; Mehta et al, 2004;

Rowe et al, 1974; Stryker & Kreps, 2001). Achalasia (esophageal motility disorder that can

result in gastric reflux) and jejunal diverticulosis (able to cause small bowel perforation) can

also occur (Banikazemi et al, 2005). These clinical manifestations are thought to be related to

neuropathy of the autonomic nervous system and deposition of GSLs in ganglia and small

mesenteric vessels (Hoffmann et al, 2004).

Neuropathic pain is present very early in FD patients and is generally the first clinical

manifestation of the disease. However, the peripheral somatic and autonomic nerve systems

are already affected in childhood and 41-59% of the FD children from both genders feel pain

(Cable et al, 1982; Dutsch et al, 2002). In adolescence, the neuropathic pain experience

increases to 65-81% among FD patients (Hopkin et al, 2008). Pain can be characterized by

episodic crises of acute burning pain at the extremities or chronic paraesthesias, triggered by

physical or emotional stress (Hoffmann et al, 2007). The physical limitations resulting from this

symptom significantly reduce the patient’s quality of life (Germain, 2010).

1.5.1. Heterozygotes

For many years the FD females were seen as carriers of the disease, showing few or

none of the clinical symptoms. However, the number of case reports including females

showing signs and symptoms of FD, continued to increase, and large surveys among patients

with FD revealed that there was a high prevalence of affected females (Figure 7; Desnick et al,

2001; Mehta et al, 2004; Bodamer, 2007). Since α-Gal A activity in FD females is often within

the normal limits even in cases of severe clinical phenotype, it is difficult to elaborate the

diagnosis, and therefore a combination of screening techniques including enzyme activity

assays, measurement of urinary substrate excretion and molecular analysis, has to be carried

out (Bodamer, 2007).



[15]

The most common complications experienced by FD heterozygous females are

acroparesthesia, present in more than 80% of these patients since childhood (MacDermot et al,

2001b; Whybra et al, 2001), angiokeratoma, found in more than 55% of FD females (Whybra

et al, 2001), renal impairment, affecting up to 50% of the female patients (MacDermot et al,

2001b; Wang et al, 2007) and cerebrovascular accidents, in 5-21% of the females (Fellgiebel et

al, 2006; MacDermot et al, 2001b; Mehta et al, 2004; Rolfs et al, 2005). Heart manifestations

were also found in some affected females, like hypertrophy, cardiomyopathy, cardiac valve

disease, and arrhythmias (Chimenti et al, 2004; Elleder, 2010; Kampmann et al, 2002; Mehta

et al, 2004; Shah et al, 2005).

Figure 7: Reported signs and symptoms of Fabry disease among 366 FD patients. For this study it was considered

the age of the FD patients at the time of entry into the European Fabry Outcome Survey (FOS) in hemizygous males (black bars)

and heterozygous females (white bars). (Mehta et al, 2004).



[16]

The lifespan of FD females is therefore reduced upon these complications and efforts

must be made concerning their diagnosis, since many of the heterozygous females are still

misdiagnosed.

1.6. Treatment

ERT is available for FD patients since 2001 in Europe in the form of two different

drugs, Agalsidase alfa (Replagal®, Shire Human Genetic Therapies), and Agalsidase beta

(Fabrazyme®, Genzyme Corporation; Lidove et al, 2010).

Both Agalsidase alfa and beta are designed to boost α-Gal A activity. The amino acid

composition of Agalsidase alfa and Agalsidase beta is the same and both are capable of

successfully correct the substrate storage in FD patients cultured fibroblasts (Blom et al,

2003). On the other hand, differences in glycosylation and the number of mannose-6-

phosphate residues have been described (Lee et al, 2003; Sakuraba et al, 2006). Agalsidase

beta possesses more mannose-6-phosphate residues than Agalsidase alfa and the number of

mannose-6-phosphate will influence the target compartment of these enzymes within the cell,

which has to be the lysosome in order to fulfill its role. (Lee et al, 2003; Sakuraba et al, 2006).

Since the glycosylation process might be important for enzyme input in human cells, and once

glycosylation in Agalsidase beta is from non-human cells, it is possible that IgG antibodies are

produced against this drug (Eng et al, 2001; Schiffmann et al, 2001). Nevertheless, the

production of antibodies can be stimulated by both enzymes, with higher probability in patients

with lower α-Gal A activity (Eng et al, 2001; Schiffmann et al, 2006). These antibodies

neutralize the enzyme activity (Ohashi et al, 2007). Studies made on male patients being

treated by ERT show that Agalsidase alfa is less antigenic than Agalsidase beta and therefore

less antibodies are produced against this enzyme (Vedder et al, 2007a).

The approved dose of enzyme is 0.2 mg/Kg for Agalsidase alfa and 1.0 mg/Kg for

Agalsidase beta every 15 days. Both treatments have variable results in FD patients, but

preservation and stabilization of the renal function has been described, as well as reduction of

cardiac hypertrophy, infarct and diarrhea episodes, fatigue and pain (Connock et al, 2006).

Hearing loss, ‘psychological status’, frequency of physical exercise, ability to sweat and quality



[17]

of life were improved in treated patients (Connock et al, 2006; Wilcox et al, 2004). However

ERT cannot stop FD from evolving if it is already in an advanced stage, especially when renal

insufficiency is already present (Vedder et al, 2007a). For this reason, it is extremely important

to start the therapy as soon as possible, in order to significantly improve lifespan of FD

patients. An alternative to the ERT is Substrate Reduction Therapy (SRT), which inhibits the

GSL biosynthesis in order to reduce accumulation of the α-Gal A substrates (Platt et al, 1994).

This therapy was only tested in the animal model of FD and lymphoblasts of FD patients (Abe

et al, 2000a; Abe et al, 2000b; Heare et al, 2007).

Although SRT and ERT can both reduce the storage substances, a recent study

combining both therapies in FD mice revealed that SRT works as an adjuvant for ERT,

improving the beneficial effects in many tissues, where higher storage clearance results were

obtained (Marshall et al, 2010).

1.7. Laboratory Diagnosis of Fabry Disease

The Fabry outcome survey, a survey of a large cohort of Fabry patients, revealed that

the average age for the FD diagnosis was 25.7 ± 15.3 years and 31.3 ± 17.4 years for male

and female patients, respectively, with an average delay between onset of symptoms and

diagnosis of approximately 12 years for both genders (Beck, 2006). Although the clinical

history raises the suspect of FD, the definitive diagnosis in male patients is done when

diminished α-Gal A activity is detected in a blood sample (Caudron et al, 2007; Desnick et al,

2001; Winchester & Young, 2006). The blood sample can either be leukocytes, plasma/serum

(Winchester & Young, 2006) or DBSs (Chamoles et al, 2001; Gaspar et al, 2010). α-Gal A

activity can also be determined in cultured fibroblasts, urine and other tissues (Winchester &

Young, 2010). To confirm the diagnosis, mutational analysis of the GLA gene can be

performed. In heterozygous females, the measurement of the α-Gal A activity is not sufficient to

give a reliable diagnosis since the α-Gal A activity may be within the normal range, due to

random inactivation of the X-chromosome (Germain, 2010). Hence, female samples must be

submitted to mutation analysis as well.



[18]

The accumulation of α-Gal A substrates, namely Gb3 and lyso-Gb3, in plasma and

urine can be measured and used to support the diagnosis and for treatment monitoring (Aerts

et al, 2008; Eng et al, 2001; Togawa et al, 2010).

The described methods for direct Gb3 identification and measurement are thin-layer

chromatography (TLC; Berná et al, 1999; Philippart et al, 1969), enzyme-linked

immunosorbent assay (ELISA; Zeidner et al, 1999) and mass spectrometry (MS; Domon &

Costello, 1988; Hsu & Turk, 2001; Mills et al, 2002). Other methods include

immunohistochemistry (Selvarajah et al, 2011), indirect Gb3 identification by release of the

oligosaccharide moiety by gas-liquid chromatography (GLC; Vance & Sweeley, 1967) or high-

performance liquid chromatography (HPLC; McCluer et al, 1989; Ullman & McCluer, 1977).

The ultimate efforts made to improve the screening methodologies concern the mass

or high-risk screening, based on measurement not only of α-Gal A activity and/or protein in

DBSs, but also of the storage products in urine collected on filter paper. Some screening

programs for FD based on MS measurement of Gb3 levels in urine and plasma, produced

satisfactory and relevant results for comprehension of disease severity (Auray-Blais et al, 2007;

Auray-Blais et al, 2006; Boscaro et al, 2002; Chien et al, 2011; Kitagawa et al, 2005). On the

other hand, MS is a time-consuming method that is economically and technically very

demanding, requiring specialized data interpretation.

TLC is a classic method for lipid identification used over thirty years in GSL analysis.

The TLC resolving power, robustness and easy handling makes it a very reliable method for

identification and separation of neutral GSLs and gangliosides (Kundu, 1981; Müthing, 1996;

Müthing, 1998; Schnaar & Needham, 1994; Skipski, 1975; Yu & Ariga, 2000). Thus, it is a

method suitable for a mass screening program.

The diagnosis of FD must be done as early as possible to achieve a better prognosis.

However, the newborn screening of late onset X-linked disorders such as FD isn't generally

accepted. For this reason, the screening of high-risk populations, which are composed of

individuals with family history of FD or have symptoms compatible with FD, is the most

adequate approach.

In fetus, the diagnosis can be achieved through the assessment of α-Gal A activity in

the fetal tissues, since it reflects the genotype of the fetus. Hemizygous males are diagnosed

due to deficient α-Gal A activity and heterozygous show variable amounts of activity. In male



[19]

fetus, the diagnosis can be made directly by measuring α-Gal A activity and/or mutation

analysis (if the family mutation is known) directly in the chorionic villi and in cultured chorionic

villi and amniotic cells (Winchester & Young, 2010). The female fetus can only be reliably

diagnosed by mutational analysis, however it is not possible to predict what exactly will be the

course and severity of FD in the heterozygous females for a certain mutation (Beck, 2006).

CHAPTER II

2. STUDY AIMS



[20]

FD is a lysosomal storage disorder originated by a deficient activity of α-Gal A caused

by mutations in the GLA gene. The substrates hydrolyzed by α-Gal A, such as Gb3 and other

related GSLs, accumulate in different tissues and organs causing its dysfunction and are

usually excreted in urine.

FD is an X-linked disease, however it is known that not only the males but also most of

the females heterozygous for GLA mutations, present symptoms, although FD female patients

have a late onset and show a slower progression of the disease. The severity and rate of

progression of the disease is dependent on the degree of residual enzyme activity. The males

with a classic form of FD are easy to diagnose since they present no residual activity, on

contrast to females (heterozygous) and the males with a non classic form of FD, as they

present some residual activity, and the diagnosis implicates combined biochemical and genetic

analyses.

ERT is available for FD patients in the form of Agalsidase alfa and Agalsidase beta,

both showing multi-organ beneficial effects. However, it must be noticed that the earlier the

treatment starts, the higher are the chances of increasing the lifespan and quality of life of

these patients.

Many studies focus on the diagnosis of FD by measuring the α-Gal A activity in blood

and accumulation of its substrates, mainly Gb3, in urine and plasma of high risk populations.

Mass spectrometry is widely applied for the screening of FD, as well as of other LSDs.

However, mass spectrometry is a very expensive method, requires much skill from the

operator and involves complex equipment. TLC is a robust and well explored method, suitable

to analyse a large number of samples.

Urine samples are easy to collect, facilitating the cooperation of individuals, but on the

other hand, the transportation is difficult without compromising the samples' integrity.

Thus, this study aims to use urine samples of FD patients to define a reliable diagnosis

method, applicable to an automatic screening program of FD, and to follow the patients that

are on ERT.

Therefore, this study has the following specific aims:

1. Simplification of the shipping method of urine samples using the filter paper as

support material.



[21]

2. Optimization of the analysis method of Gb3 used in the laboratory for analysis

of filter paper urine samples.

3. Obtain a screening system able of discriminating FD patients (males and

females) from healthy individuals, and useful to follow FD patients under

treatment.

4. Determination of the GSL profile in urine of FD patients, with known mutations

of the GLA gene, before and after the beginning of the ERT.

CHAPTER III

3. SAMPLES AND METHODOLOGY


Chapter III – Samples and Methodology

[23]

3.1. Sampling

3.1.1. Biological samples

For this study eleven FD patients and thirty control subjects were analysed. The

samples from control subjects were obtained from internal volunteers with representation of

both genders and with ages between 20 and 69 years (Table II and Appendix I – Table III). Of

the thirty control subjects 23 samples were collected with the filter paper kit. As for the FD

patients, samples were obtained through a protocol established with the physicians and

hospitals directly linked to the patients1. Samples from FD patients are from both genders and

their ages are between 2 and 67 years (Table IV). Due to the established protocols, all patient

samples were conventionally collected. The GLA gene mutations were previously identified and

the mutations C94S, F113L, M290I and R118C in the studied patients. Only two of the

patients presented samples at least in two time points during ERT.

Table II: Cohort characteristics. Description of patient (n=11) and control subjects (n=30) regarding their age, collection

method (filter paper kit or conventional liquid collection) and gender. Age is presented as mean. The age range is displayed in

parenthesis.

Samples Age Collection Method Gender

Filter Paper Conventional Males Females

Patients (n=11)

31.4 (2-67)

0 11 8 3

Controls (n=30)

37.9 (20-69)

23 7 15 15

1 Protocols established between Dr. Olga Azevedo – Centro Hospitalar do Alto Ave, Dr. Elisa Leão – Hospital S. João, Dr. Célia Barbosa – Hospital Pedro Hispano and Dr. Miguel Rodrigues – Centro Hospitalar de Setúbal.



[24]

Table IV: Characteristics of FD patients. Discrimination in terms of gender, age, GLA gene mutation and treatment

duration at the time of sample collection. M – male; F – female. Asterisk (*) represents family related patients.

Patient Gender Age GLA Mutation Treatment time at sample collection

(months)

1 M 9 C94S 0

2 M 40 F113L 0, 3, 8

3 M 49 F113L 0

4 M 24 F113L 0

5 M 2 M290I 0

6 F 44 M290I 0

7* F 35 F113L 0

8 M 11 F113L 0

9* M 18 F113L 0

10 M 47 R118C 0

11* F 67 F113L 5, 17

3.1.2. Sample Collection

Liquid urine samples were collected in the flask seen in Figure 8, aliquoted and frozen

at -20 ºC until the analysis. Filter paper urine samples were collected using the kit in Figure 9.

The recipient carried two filter paper squares, each one of 5 x 5 cm and was accompanied by

an envelope containing a forceps. The donor was instructed of how to collect the sample and

handle the filter papers through an instructions sheet (Appendix I – Figure 10). The collection

indications were as follows: 1) dispose the excess of urine after absorption by the filter papers;

2) dry the filter papers at room temperature; 3) use the forceps to handle the dried filter

papers and send them by mail using the provided envelope. At the arrival, the samples were

catalogued and stored at -20 ºC in a sealed bag.



[25]

Figure 8: Conventional collection urine flask. Urine collection flask with capacity to 100 mL.

Figure 9: Filter paper kit. A. Urine collection container of 200 mL with two 5 x 5 cm filter papers (Whatman® #903). B.

Filter paper kit for urine collection composed by one urine container with filter papers, one envelope and one sealed forceps.

All our patient samples were 20 mL conventionally collected urine samples stored at -

20 ºC. For this matter, these samples had to be defrosted and applied to filter papers, 2 mL

per filter paper.

3.2. Reagents and Standards

Organic solvents such as chloroform (CHCl3) and methanol (MeOH; Fisher Scientific®

HPLC grade) were used to make organic solvent solutions or for single use.

The derivatization solution was produced with p-anisaldehyde (Sigma Aldrich®) and 96%

PA sulphuric acid (H2SO4; Panreac®).



[26]

Several glycosphingolipid standards were used to prepare standards for the HPTLC

plates, to spike and overload samples. The standards were SM, Gb4q, Gb3, LacCer, GlcCer

and phosphatidylethanolamine (PE) from Matreya®.

3.3. Methods

In order to carry out the proposed aims, blood and urine samples collected on filter

paper were analyzed. High-Performance Thin-Layer Chromatography (HPTLC), was used to

identify traceable amounts of GSLs excreted in urine, namely Gb3. This technique was

optimised to improve its sensitivity and to decrease its time duration for a better discrimination

of FD patients and control subjects. This part was developed within the screening project for

FD from Instituto de Biologia Molecular e Celular – Instituto de Engenharia Biomédica (IBMC-

INEB), in Portugal, fellowship PTDC/SAL-BEB/100875/2008. The obtained results were also

integrated in the IBMC-INEB project. The INEB team is currently developing an automatic

screening software.

3.3.1. High-Performance Thin-Layer Chromatography for

Conventional Urine Samples

Liquid urine samples are dialyzed in 12-14 kDa dialysis membrane against 0.01%

sodium azide and dried by lyophilization.

Lipids are then extracted in two cycles with centrifugation: one cycle of 1min vortexing

with 20:1.5 (v/v) MeOH/H2O and CHCl3 and one cycle of 1 min vortexing with 1:2 (v/v)

CHCl3/MeOH, followed by concentration under N2 stream, at 40 ºC.

The total lipid extract is then fractionated in C18 chromatography columns

(Chromabond® C18 octadecyl-modified silica columns). Neutral lipids are recovered by a 1:2

(v/v) CHCl3/MeOH solution. The existing contaminants are removed and the acidic lipids are

discarded. In order to remove phosphoglycerides, neutral lipids are subjected to alkaline

hydrolysis with sodium hydroxide. After hydrolysis, dried fractions are reconstituted in 1:2 (v/v)

CHCl3/MeOH and applied to an HPTLC silica gel plate (Merck® HPTLC silica gel 60 plates)

using a spray-on sample application device (Camag® Linomat IV). To allow the identification



[27]

and semi-quantification of the lipids on each sample, a mixture of standard lipids incorporating

known amounts of SM, Gb4, Gb3, LacCer, GlcCer and PE is applied (Table V). Plates are

developed with 70:30:5 (v/v) CHCl3/MeOH/H2O in a TLC chamber (Camag®) and dried under

a flow of hot air. The resulting separated lipids are visualized as purple bands by pulverizing

the plate with an p-anisaldehyde/ H2SO4 solution (1% p-anisaldehyde in 80% H2SO4) followed by

heating the plate in the oven at 120ºC until the appearance of bands. Plates are digitalized and

bands are quantified by the UN-SCAN-IT gel automated digitizing system software version 5.1

(Silk Scientific corporation).

Table V: Constitution of a standard mixture for HPTLC. 30 μL 0.2 μg/μL disposable mixture aliquots.

GSL Quantity per 10

μL

GlcCer 2 µg

PE 2 µg

LacCer 2 µg

Gb3 2 µg

SM 2 µg

Gb4 4 µg

This procedure will allow the classification and semi quantification of the lipids present

in each sample, allowing a rapid screening of significant alterations in the lipid profile as

compared to control samples.

CHAPTER IV

4. RESULTS


Chapter IV – Results

[29]

4.1. High-Performance Thin-Layer Chromatography Optimization

for Filter Paper Urine Samples

4.1.1. Validation of Filter Paper for HPTLC Analysis

Liquid urine samples are difficult to send to the laboratory and only are viable for

several hours before the storage at -20 ºC. On the other hand, filter papers can be sent by mail

and are viable for several weeks even at room temperature (Auray-Blais et al, 2007). However,

the HPTLC method was not adapted to filter paper collected samples. The main problem was

the significantly lower urine quantity held by the filter paper when compared to the

conventionally collected urine samples. In order to do the screening for FD, 20 mL of urine are

needed, but the filter paper squares (5 x 5 cm) only absorb a maximum of 2 mL.

To evaluate if the use of 1filter paper is a suitable approach, the same urine sample

was added to one filter paper and two filter papers (approximately 4 mL; Figure 11). The Gb3

of each sample was normalized to SM values. SM is an adequate physiological representative

of Gb3 excretion, as like Gb3, it can be localized at the cellular membrane (Barr et al, 2009).

Because the SM and Gb4 bands usually overlap they are identified as a group.

It is visible that in the urine samples applied to two filter papers the GSL bands are

stronger, when compared to samples applied to one filter paper (confirmed in Table VI). The

ratio of Gb3 to SM+Gb4 of the one filter paper sample overloaded with Gb3 proved to be higher

than the one from the two filter papers, not because of Gb3 bands intensity but because there

was less SM quantified, in similarity to the non-overloaded samples.

Analyzing the Gb3 band in overloaded samples it is also clear that the use of two filter

papers does not interfere with Gb3 identification and band density. Therefore, in order to

increase the amount of GSLs in urine samples, the minimum sample quantity was defined as

two filter papers.



[30]

Figure 11: Analysis of 1 and 2 filter papers from a control sample. An urine sample from a control subject was

applied to filter papers and analysed by the conventional HPTLC method. The same sample was divided into: 1) 2 filter papers,

2) 1 filter paper, 3) 1 filter paper overloaded with 2 µg of Gb3, and 4) 2 filter papers overloaded with 2 µg of Gb3. F.P. – filter

paper. Std. – standards (2 µg). The applied standard mixture contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of

Gb4.

Table VI: Densitometry of Gb3 and SM+Gb4 bands from the filter paper samples. The SM/Gb4 and the Gb3 bands

from the samples of the control subject previously analysed by HPTLC were quantified by densitometry. SM/Gb4 group was

analysed separately of Gb3. The ratio of Gb3 to SM+Gb4 was calculated to normalize the samples. The represented pixels are

the result of the background pixels subtracted to the total band determined pixels. 2 F.P. – two filter papers of the urine

sample; 1 F.P. – one filter paper of the urine sample; 1 F.P. + Gb3 – one filter paper of the urine sample overloaded with Gb3;

2 F.P. + Gb3 – two filter papers of the urine sample overloaded with Gb3. Gb3 overload – 2 µg; n.d. – non-detected.

Lane Gb3

(pixels) SM+Gb4 (pixels)

Gb3/SM+Gb4

(pixels)

2 F.P. 77.1 497.9 0.15

1 F.P. n.d. 349.3 n.d.

1 F.P. + Gb3 82.0 355.7 0.23

2 F.P. + Gb3 92.4 514.6 0.18

This change was tested in several naïve patient samples (not on ERT; Figure 12).

Although the filter paper Gb3 bands were markedly weaker compared to the ones obtained



[31]

from liquid samples, the FD patients detected in liquid samples were also detected by filter

paper analysis. Moreover, the software that is currently being developed must be sensitive

enough to discriminate FD patients from controls.

Figure 12: FD patients’ analysis of conventional 20 mL urine samples and urine samples applied to filter

papers. 20 mL urine samples from previously diagnosed FD patients (P1-P4) were analysed by the conventional HPTLC

method prior to this study. The same samples were added to two filter paper squares and analysed by the optimised HPTLC

method that is later described in this work2. Std. – standards (2 µg). The applied standard mixture contained 2 µg of SM, Gb3,

LacCer, PE and GlcCer and 4 µg of Gb4.

4.1.2. Extraction Method

The extraction method limits the lipid quantity available for the following HPTLC steps.

The mechanical procedures (centrifugation, sonication and agitation) and the organic solvent

solutions were the tested conditions.

The centrifugation steps are crucial for protein precipitation. However, the filter paper

was described as a good support for proteins since they do not enter in solution during the

extraction process (Müthing & Distler, 2009). It has also been suggested that the desquamated

urinary tract cells may still be intact in the urine extract and that sonication improves the

amount of detected lipid by disrupting the existing cellular and lysosomal membranes (Auray-

Blais et al, 2006). Hence, three different extraction processes were tested using a filter paper

control sample overloaded with both SM and Gb3 (Figure 13).

2 As there are few urine samples from FD patients, the filter paper analyses of all the patients

simultaneously were only performed when the method was optimised.



[32]

Figure 13: Evaluation of different extraction conditions. A control urine sample collected by the filter paper kit was subjected to three

extraction conditions, using for each one, two filter papers: centrifugation (“Cent.”) at 3000 rpm for 10 minutes (twice) after the agitation with

organic solvents; no centrifugation and no sonication steps (“no Cent./Son.”); and sonication in water bath for 5 minutes (“Son.”) of the final

extract (after the second extraction cycle). Before the extraction, each two filter paper squares were overload with 4 µg of Gb3 and 10 µg of SM.

Std. – standards (2 µg). The applied standard mixture contained 2 µg of Cer 4 (asialoganglioside GM1), 2 µg of SM, 2 µg of Gb4, 2 µg of Gb3,

4 µg of LacCer, 2 µg of PE and 2 µg of GlcCer.

The first extraction process encompasses two centrifugation steps, the second

extraction process had no centrifugation or sonication steps and the third was defined by a

sonication step. By densitometry of Gb3 bands it becomes clear that centrifugation decreases

the Gb3 quantity at the end of the extraction process. This is probably due to the extract

volume retained in the filter paper as a consequence of filter paper compression during

centrifugation (Table VII).

Table VII: Densitometry of Gb3 and SM+Gb4 bands from urine samples extracted under different conditions. The represented

pixels were determined as a result of background subtraction to the total pixels per band. Gb3 pixels and SM+Gb4 pixels were measured by

densitometry of the Gb3 and SM+Gb4 areas. Cent. – 2x 10 minutes centrifugation at 3000 rpm; no Cent./Son. – no centrifugation or

sonication steps; Son. – sonication for 5 minutes in a water bath.

Lane Gb3 pixels SM and Gb4

pixels

Cent. 1252.1 1181.5

no Cent./Son.

1817.3 1544.7

Son. 2047.9 1714.4



[33]

On the other hand, adding a sonication step increased the Gb3 band density which

indicates that more lipid was present in the extract. Although 4 μg of Gb3 were added to the

control sample, there is always some GSL content from the sample itself, producing an

increment in density relatively to the sample that was not sonicated. The densitometry of the

SM and Gb4 bands confirmed this theory.

Together, these modifications increased the efficiency of the extraction method, which

led to an improvement of the detection limit.

The type and duration of agitation during the extraction procedure is also a determining

factor for an efficient lipid extraction. In order to evaluate this factor, an empty filter paper was

overloaded with Gb3 and subjected to different agitation conditions (Figure 14A).

The standard extraction, with two cycles of organic solvents and 1min vortexing (or 5

minutes in a multi-vortex), was compared to: 1) three cycles of organic solvents, 2 h each in a

roller agitator, and 2) one cycle of 1min vortexing with organic solvents and one cycle of 24 h

agitation in a roller agitator with organic solvents. Better results were obtained with a roller

agitator when the agitation time was increased to 24 h. This observation was confirmed by

densitometry (Table VIII of Appendix II).

Another issue involves the concentration of organic solvents. The ratio between the

organic solvents is very important for an adequate extraction of the the lipids of interest. Some

authors use a 2:1 (v/v) CHCl3/MeOH ratio for GSL extraction (Krüger et al, 2010; Roy et al,

2004; Rozenfeld et al, 2009b). With the aim of exploring this variant, a filter paper control

sample was extracted with 2:1 (v/v) CHCl3/MeOH ratio and the conventional 1:2 (v/v)

CHCl3/MeOH ratio (Figure 14B). Because the control sample did not have elevated GSL

content, filter papers were overloaded with different quantities of Gb3 (2 μg and 4 μg). With the

Gb3 overload of control samples it was clear that the utilization of the 2:1 (v/v) CHCl3/MeOH

in substitution of the standard 1:2 (v/v) CHCl3/MeOH ratio led to less GSL content on the

HPTLC plate.



[34]

Figure 14: Test of different extraction agitation methods and organic solvent solutions. In A, six pairs of empty

filter papers were overloaded with 10 µg of Gb3 and extracted with: 1) one cycle of 5 minutes vortexing at room temperature

and one cycle of 24 h at 4 ºC in a roller agitator (“24 h” lanes); 2) three cycles of 2 h at 4 ºC in a roller agitator (“3 x 2h”

lanes); and 3) two cycles of 5 minutes multi-vortexing at room temperature (“5 min” lanes). In B, the same control sample

collected in filter papers was extracted with 1:2 (v/v) of chloroform/methanol (lanes 1, 3 and 5) and 2:1 (v/v) of

chloroform/methanol (lanes 2, 4 and 6), without Gb3 overload (lane 1 and 2), with 2 µg of Gb3 (lanes 3 and 4) and with 4 µg

of Gb3 (lanes 5 and 6). Analysis of two filter paper squares per lane. C/M (1:2) – 1:2 (v/v) chloroform/methanol; C/M (2:1) –

2:1 (v/v) chloroform methanol; Gb3 overload: no Gb3 overload “-“, 2 µg of Gb3 “+” and 4 µg of Gb3 “++”; Std. – standards (2

µg). The applied standard mixture contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of Gb4.



[35]

4.1.3. Lipid Fractionation Process

C18 reverse phase silica columns are specific for neutral substances. However, their

efficiency is not 100%. As the polypropylene material used for fractionation is a source of

contaminants, all the pieces preventing the sample from a direct contact with the C18 column

were removed.

To address the most adequate solvent volume to elute neutral lipids and increase the

amount of lipid eluted from the C18 column, a known quantity of Gb3 was added to a C18

column and the efficiency of the organic solvent volume used in the protocol [8 mL of 1:2 (v/v)

CHCl3/MeOH] was compared to the double volume of the same organic solvent solution

(Figure 15). Still, no improvement was seen. In fact, there was less lipid content in the lanes of

more eluted samples. The loss of lipid content was even higher in the Gb3 eluted with 16 mL

of organic solvent solution when compared to pure Gb3 lanes (Table IX).

Figure 15: Elution of neutral lipids fraction. 4 µg of Gb3 were applied to eight C18 reverse phase columns and

fractionated by the conventional method for lipid fraction collection. The only modification was the removal of the plastic

material. The neutral lipids’ fraction was eluted with 8 mL of 1:2 (v/v) CHCl3/MeOH (“8 mL C/M (1:2)” lanes), and with 16 mL

of 1:2 (v/v) CHCl3/MeOH (“16 mL C/M (1:2)” lanes). To compare the Gb3 obtained from the fractionation process to the

initially added Gb3, two lanes with 4 µg of pure Gb3 were added to the HPTLC plate (“Pure Gb3” lanes). C/M (1:2) – 1:2 (v/v)

CHCl3/MeOH; Std. – standards (2 µg). The applied standard mixture contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4

µg of Gb4.



[36]

Table IX: Densitometry of Gb3 eluted with 8 mL or 16 mL of organic solvent solution during fractionation. The

presented pixels for the Gb3 substance are discriminated from the background pixels. The following values were calculated for

pixels from the same elution condition: mean, maximum, % RSD and percentage of lost pixels relatively to pure Gb3 (“% Loss”).

C/M (1:2) – 1:2 (v/v) CHCl3/MeOH.

Lane Gb3

Mean ± SD (pixels)

Max. lane Gb3

(pixels) % RSD % Loss

8 mL of C/M (1:2)

1226.1± 157.2 1337.2 12.8 25.0

16 mL of C/M (1:2)

952.9 ± 315.7 1261.9 33.1 41.7

Pure Gb3 1635.6 ± 7.9 1641.2 0.5 0.0

The variability seen in the 16 mL eluted Gb3 may be due to insufficient reconstitution

of N2 dried GSLs during alkaline hydrolysis. As this is a high volume, GSLs will contact and

remain in areas of the material where the reconstitution solution cannot reach, preventing an

uncertain GSL quantity to be available for alkaline hydrolysis and application.

4.1.4. Development of the HPTLC Plate

The development distance is extremely important to manipulate the position and

separation of the lipids. This factor also influences the definition of the bands.

The filter paper samples have more contaminants than liquid urine samples, which

promotes the smear and diffuse aspect of lanes from filter paper samples.

A reduction in the development distance may positively influence the filter paper lanes

in what concerns to the definition. For this reason, instead of the conventional 6.5 cm of

development from the point of application (8 cm from the base of the plate), the development

was along 5.5 cm (7 cm from the base of the plate; Figure 16). Yet, no improvements were

seen with this alteration.

In order to remove most of the contaminants from the filter paper samples, a negative

control will be included in all future HPTLC plates. The automated analysis software that is

currently being developed will subtract the contaminants identified in the negative control to the

urine filter paper samples. As a consequence, the GSL bands would appear sharper.



[37]

Figure 16: Development of the HPTLC plate for 7 cm. Lipids from samples of control subjects 1 and 2 (C1 and C2)

collected in filter papers were extracted and fractionated considering the improvements described above. Each lane has the

urine quantity present in two filter papers. The alkaline hydrolysis, application in the HPTLC plate and derivatization followed the

original protocol. During the development of the HPTLC plate, lipids were separated with the 70:30:5 (v/v) of

chloroform/methanol/water solution along 5.5 cm, instead of the standard 6.5 cm. Std. – standards (2 µg). The applied

standard mixture contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of Gb4.

4.1.5. Spraying Solution

When pulverizing with p-anisaldehyde some white shadows appeared near the lipid

point of application of several plates, possibly corresponding to unstained lipids or

contaminants, as seen in Figure 17.

Figure 17: Visible white bands in patients and control urines. Three patients (P5, P6 and P7) and one control subject

(C3) were previously analysed by the original HPTLC method using 20 mL urine samples (asterisks) and samples added to two

squares of filter paper. One of the control samples had 2 µg of Gb3 added to the filter paper (“C3 + Gb3”). The white shadows

(Das et al) are visible in almost all samples in this HPTLC plate. Std. – standards (2 µg). The applied standard mixture

contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of Gb4.



[38]

This chemical compound only stains neutral lipids and for this matter, orcinol, which

stains glycolipids, was tested as a substitute for p-anisaldehyde (Figure 18).

Figure 18: Orcinol staining of neutral lipids. In A, several control samples (C1, C2, C4-C6) where analysed by

the HPTLC considering the advances in extraction and fractionation of this study. The alkaline hydrolysis, the application of

lipids in the silica plate and the separation of the lipids were executed as in the conventional method. Orcinol was the

derivatization agent. The C1, C2 and C6 were spiked with 2 µg of Gb3, the C4 and C5 were spiked with 2 µg of SM and 4 µg

of Gb4 and the C4* and C5* samples were not spiked with any GSL. In B, pure Gb4 (4 µg) and SM (2 µg) were directly applied

to the HPTLC plate. The left HPTLC plate was derivatized with orcinol and the right plate was derivatized with p-anisaldehyde.

The SM position is indicated in both derivatization methods (Das et al). Std. – standards (2 µg). The applied standard mixtures

of A. and B. contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of Gb4.



[39]

The orcinol staining was performed in control filter paper samples and lipid standards

to better understand how orcinol affects the samples and pure lipids.

As a result of this alteration the SM band disappeared and only the Gb4 band

remained, allowing the identification of Gb4 in control subjects without interference of the SM

band. Although the Gb3 is detected, the orcinol derivatization did not highlight any lipid bellow

the Gb4 band and it is less sensitive than p-anisaldehyde, when comparing the orcinol stained

samples of Figure 18 to the p-anisaldehyde stained samples of Figure 17. Furthermore, the

SM/Gb4 conjugation was pointed as necessary to adjust the parameters of the automatic

screening software. For these reasons the orcinol staining could not be adopted.

4.1.6. Gb3 Detection Limit

In order to evaluate the detection limit of Gb3 in the HPTLC plate, an overload study

was made (Figure 19). Several pairs of empty filter papers were overloaded with increasing

concentrations of Gb3 (0.5 µg to 4.0 µg). As a result, the existence of Gb3 can only be

guaranteed if the amount of this lipid in the HPTLC plate is higher than 2 µg.

Moreover, a high quantity of Gb3 is still being lost during the HPTLC as the 4 µg

overloaded filter paper bands cannot surpass the Gb3 density of the standard mixture (with 2

µg of Gb3).

Figure 19: Estimation of the Gb3 detection limit. Empty filter papers were grouped in pairs, to assure that the same quantity of

impurities observed in the urine samples was present, and subsequently overloaded with Gb3 (0.5 µg to 4.0 µg). Filter paper Gb3 was analysed

by HPTLC regarding all the modifications established so far. Std. – standards (2 µg). The applied standard mixture contained 2 µg of SM, Gb3,

LacCer, PE and GlcCer and 4 µg of Gb4.



[40]

4.1.7. Lipid loss in the HPTLC technique

Besides the improvement in resolution of GSL bands, some lipid content is lost along

the HPTLC method, mainly during extraction and fractionation.

In order to determine the percentage of Gb3 loss in the whole HPTLC method, 4 µg of

Gb3 where added to an empty filter paper and subjected to the complete optimised method

(Figure 20A). As part of the lipids are lost during fractionation, 4 µg of Gb3 where directly

added to a C18 column without being extracted. The application of 4 µg of Gb3 directly to the

HPTLC plate, guarantees that there is no lipid loss and can therefore be considered as 0% loss

and used to compare the other lanes. Both filter paper Gb3 and fractionated Gb3 suffered

alkaline hydrolysis, which means that the differences between these Gb3 bands are almost

exclusively attributed to the extraction and fractionation process.

Figure 20: Gb3 loss during the HPTLC technique and fractionation of the optimised and conventional method.

A. Optimised method; B. conventional method. Both plates followed the following principle: two empty filter papers were

overloaded with 4 µg of Gb3 (“F.P. + Gb3”) to determine the total loss of Gb3 during the technique. 4 µg of Gb3 were added to

a C18 chromatography column fractionated (“Column + Gb3”) in order to determine the Gb3 lost during GSL fractionation. 4

µg of Gb3 were directly applied to the HPTLC plate (“Gb3”) to allow comparison. Std. – standards (2 µg); The standard mixture

in A. contains 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of Gb4. The standard mixture in B. contains GlcCer 2 µg of

Cer 4, 2 µg of Gb4, 2 µg of SM, 2 µg of Gb3, 4 µg of LacCer, 2 µg of PE and 2 µg of GlcCer.



[41]

By densitometry it was possible to quantify the decrease of both filter paper Gb3 and

fractionated Gb3 relatively to the directly applied Gb3, which was of 37.5% and 25%,

respectively (Table X). This means that approximately 12.5% of Gb3 is lost during extraction.

The same experiment was performed using the conventional HPTLC protocol (Figure

20B). With the conventional method, the filter paper Gb3 also demonstrates less dense bands

and some Gb3 is lost in fractionation. The quantification of this loss was done by densitometry

and shows that 51.1% of Gb3 is lost with this HPTLC method and 29.7% of Gb3 is lost in

fractionation. Hence, approximately 21.4% of Gb3 is lost in the extraction process.

Table X: Densitometry for calculation of Gb3 loss during the HPTLC technique and fractionation with the

optimised and conventional methods. The Gb3 pixels were calculated as subtraction of background pixels to the total

pixels at the Gb3 area. The percentage of Gb3 loss was calculated through the Gb3 pixels relatively to the directly applied Gb3

(considered 0% loss). The added and applied Gb3 quantity was 4 µg.

Comparing both methods, it is clear that most Gb3 is lost during the fractionation

process and that the HPTLC sensitivity has been improved by 13.6% comparatively to the

conventional HPTLC method. Therefore, the optimization of the standard HPTLC protocol for

urinary lipids resulted in the filter paper enhanced method described below.

4.1.8. Optimised Protocol

Lipids are extracted in two cycles: one cycle of 1 minute vortexing with 20:1.5 (v/v)

MeOH/H2O and CHCl3 and one cycle of 24 hour agitation with 1:2 (v/v) CHCl3/MeOH, followed

by 5 minutes sonication and concentration under N2 stream, at 40 ºC.

Optimised Method Conventional Method

Lane Gb3

(Pixels) % Gb3 Loss

Gb3 (Pixels)

% Gb3 Loss

F.P. + Gb3

592.4 37.5 845.2 51.1

Column + Gb3

711.1 25.0 1215.7 29.7

Gb3 948.0 0.0 1729.6 0.0



[42]

The total lipid extract is then fractionated in C18 chromatography columns without the

fractionation plastic materials (Chromabond® C18 octadecyl-modified silica columns). Neutral

lipids are recovered by a 1:2 (v/v) CHCl3/MeOH solution. The existing contaminants are

removed and the acidic lipids are discarded. In order to remove phosphoglycerides, neutral

lipids are subjected to alkaline hydrolysis with sodium hydroxide. After hydrolysis, dried

fractions are reconstituted in 1:2 (v/v) CHCl3/MeOH and applied to an HPTLC silica gel plate

(Merck® HPTLC silica gel 60 plates) using a spray-on sample application device (Camag®

Linomat IV). To allow the identification and semi-quantification of the lipids on each sample, a

mixture of standard lipids incorporating known amounts of SM, Gb4, Gb3, LacCer, GlcCer and

PE is applied (Table IV). Plates are developed with 70:30:5 (v/v) CHCl3/MeOH/H2O in a TLC

chamber (Camag®) along 8 cm (from the base of the plate) and dried under a flow of hot air.

The resulting separated lipids are visualized as purple bands by pulverizing the plate with an p-

anisaldehyde/ H2SO4 solution (1% p-anisaldehyde in 80% H2SO4) followed by heating the plate in

the oven at 120 ºC until the appearance of bands. Plates are digitalized and bands are

quantified by the appropriate software.

The reproducibility of this method was tested by subjecting the same sample to the

optimised method in different days (Figure 21).

Figure 21: Reproducibility evaluation of the optimised method. Several filter papers of the same control sample

(obtained through the filter paper collection kit) were grouped in pairs and analysed by the optimised HPTLC method in three

different days. Std. – standards (2 µg). The standard mixture is composed by GlcCer (2 µg), PE (2 µg), LacCer (4 µg), Gb3 (2

µg), SM (2 µg), Gb4 (2 µg) and Cer 4 (2 µg).



[43]

Table XI: Assessment of variability of the optimised method by densitometry. The SM/Gb4 pixels were calculated as

a group by subtracting of background pixels to the total pixels of the SM/Gb4 area. SM/Gb4 values are expressed as mean ±

SD. The variability of the analysis was calculated as %RSD.

Lane SM/Gb4

Mean ± SD (pixels)

% RSD

Day 1 91.3 ± 7.6 8.3

Day 2 176.5 ± 17.6 10.0

Day 3 116.3 ± 13.0 11.2

Total 123.9 ± 34.6 28.0

In the HPTLC plate only slight differences are seen in the SM and Gb4 bands, however,

the calculated RSD of 28.0% shows that some variability is inherent (Table XI). In the intra-day

variability results, lower %RSD values were achieved. Repeating this reproducibility test by a

different operator and assessing the intra-day precision should help to clarify if this degree of

variability is acceptable. Once the method variability is established, this method should be

considered reproducible.

4.2. Lipidic Profile of FD Patients and Control subjects

Obtaining and comparing the lipid profile of FD patients and control subjects is crucial

for future distinction of these two groups by HPTLC.

For this purpose, both patients and control subjects were analysed by the optimised

HPTLC method. As it is visible in Figure 22A and 22B both control subjects and FD patients

are heterogeneous. There is presence/absence of Gb3 band in the control and patient groups

and high variability is seen in Gb3 and SM+Gb4 density (78.2% RSD in control subjects and

125.0% RSD for FD patients; Table XII and Appendix II – Table XIII). However, a healthy

individual typically shows a weak band of Gb3. On the other hand, a classic FD patient

presents a strong Gb3 band.



[44]

Figure 22: Analysis of samples from control subjects and FD patients by the optimised HPTLC method. A.

Samples from control subjects (C8-C29), males and females, were collected using the filter paper kit (two filter papers each)

and then analysed by the HPTLC optimised method. B. Samples from FD patients (P1-P10), males (right plate) and females

(left plate), were added to a pair of filter papers each and then analysed by the optimised HPTLC method. FD patients carried

the following mutations: M290I (P5 and P6), F113L (P2, P3, P4, P7, P8 and P9), C94S (P1) and R118L (P10). B. Std. –

standards (2 µg). The applied standard mixture contained 2 µg of SM, Gb3, LacCer, PE and GlcCer and 4 µg of Gb4.

The densitometry analysis of Gb3 and SM+Gb4 bands from control subjects indicated

that almost all subjects had no traceable Gb3 levels in filter paper urine samples, in contrast to

urinary Gb3 levels of FD patients, where 6/10 patients presented visible Gb3 bands.

Concerning the genotype of these FD patients the following missense mutations are

represented: M290I, F113L, C94S and R118L. The F113L mutation is associated with the

cardiac variant of FD (Eng et al, 1997). From the six patients with the F113L mutation



[45]

(patients 7, 3, 4, 2, 8 and 9), only patient 8 did not show a Gb3 band. The densitometry of the

SM+Gb4 bands of this patient revealed very low SM+Gb4 levels, which may also be related to

the lack of Gb3 since SM is an indicator of the GSL quantity excreted in urine. Patients 7 and 9

are from the same family and presented tenuous Gb3 bands that would make it difficult to

discriminate these patients among a series of control subjects just by looking at the HPTLC

plate. By densitometry it is clear that although patient 7 is borderline, patient 9 would be

detected by the software analysis. The other three patients with F113L mutation (patients 2, 3

and 4) were unequivocally identified.

Table XII: Densitometry of control and FD patients with traceable amounts of Gb3. The GSL pixels were calculated

by subtraction of background pixels to the total pixels of the area with GSL bands. The ratio of Gb3 to SM+Gb4 was calculated

to normalize the samples. The mean is expressed with the SD. The variability within the control and patient samples was

assessed by % RSD. Gb3 was normalized to SM+Gb4 bands.

Sample SM+Gb4 (pixels)

Gb3 (pixels)

Gb3/SM+Gb4 (pixels)

C21 401.4 176.4 0.4

C28 246.2 88.6 0.4

Mean ± SD 323.8 ± 109.7 132.5 ± 62.0 0.4 ± 0.1

% RSD 33.9 46.8 14.0

P7 446.0 154.7 0.3

P3 75.4 403.5 5.4

P4 42.8 822.4 19.2

P2 84.7 372.9 4.4

P1 43.4 512.3 11.8

P9 31.5 124.5 4.0

Mean ± SD 120.6 ± 160.7 398.4 ± 256.1 7.5 ±6.8

% RSD 133.3 64.3 91.0

The M290I mutation was present in patient 6 (female, 44 years) and patient 5 (male,

2 years), both showing absence of Gb3, despite that M290I is described as a causal mutation

of the classic form of FD (Shabbeer et al, 2006). One possible explanation is that the random

X-inactivation that patient 6 suffered allowed the expression of the wild type α-Gal A in enough



[46]

quantity to guarantee the normal Gb3 clearance. As for patient 5, due to its young age it was

expected that Gb3 was not excreted in traceable amounts.

The Gb3 bands were absent in patient 10, which was further confirmed by

densitometry. This patient presents a R118C mutation described for its association with a late

onset form of FD (Gaspar et al, 2010; Spada et al, 2006).

The only patient with a C94S mutation (patient 1) showed a strong Gb3 band and

increased LacCer/PE bands. By densitometry analysis, this patient was identified as the

patient with higher urinary Gb3 levels, considerably above the control subjects with Gb3

excretion. The increased excretion of LacCer is described in some FD patients, including

heterozygous patients (Fuller, 2005). In the literature, the C94S mutation is associated with a

severe FD form (Blaydon et al, 2001).

In order to study the excretion levels, the population of FD patients must continue

being studied. This will allow the establishment of a range for the Gb3/SM+Gb4 ratio in urine

filter paper samples of FD patients.

4.3. Effect of ERT in FD Patients

In order to evaluate the effect of ERT in FD patients it is necessary to establish a

connection between the patient status before and after the treatment takes place.

Patient 2 had a urine sample collected before ERT, one sample after three months of

therapy and one sample after eight months of therapy (Figure 23). Patient 1 only had urine

samples collected after five months and seventeen months of therapy.

Although no differences are seen in samples from patient 11, in patient 2 it is visible

that all Gb3 is cleared from the urine after three months of therapy. This status is maintained

until he reaches eight months of therapy.

A 20 mL sample of patient 11 collected before treatment had almost no Gb3 and that

reason it was expected that ERT would clear the urine Gb3 before five months of therapy.

These results also show that the optimised HPTLC method is reliable to follow up

patients under ERT.



[47]

Figure 23: Analysis of samples from FD patients collected before and during ERT. The therapy duration at the time

of sample collection (T) was defined in months. TO represents samples collected before the beginning of ERT. Urine samples

from patient 11 (P11) and patient 2 (P2) were collected in several time-points of therapy (T5 and T17 in P11 and T0, T3 and

T8 in P2) and applied to a pair of filter papers each. The sample analysis was performed by the optimised HPTLC method. Std.

– standards (2µg). The standard mixture is composed by GlcCer (2 µg), PE (2 µg), LacCer (4 µg), Gb3 (2 µg), SM (2 µg), Gb4

(2 µg) and Cer 4 (2 µg).

CHAPTER V

5. DISCUSSION


Chapter V – Discussion

[49]

Over the years, TLC has been used as a reliable method for the identification of lipid

biomarkers, weakly polar and non-polar substances, and for the screening the various diseases

(Auray-Blais et al, 2007; Desnick et al, 1970; Philippart et al, 1969; Togawa et al, 2010;

Wherrett, 1967). The TLC technique can easily be adapted to the study of any biological

sample or specific lipid.

Mass spectrometry has been used in several screening programs for Fabry disease as

a sensitive method for Gb3 and lyso-Gb3 quantification, but on the other hand, it is expensive

and labour-intensive. Like TLC, mass spectrometry requires human interpretation and data

treatment. An error in the results’ treatment process may then create untrue results. This

disadvantage can be overcome in TLC by an automatic classification method for FD

biomarkers, namely Gb3. Moreover, a large number of samples can be classified

simultaneously.

In this laboratory, the HPTLC method was explored as a complementary method for

diagnosis and research of Fabry and Gaucher diseases using urine and plasma samples.

Plasma and urine samples are the most frequently used samples to study Gb3 accumulation in

FD populations and both produce enlightening results (Boscaro et al, 2002). As the urine

collection method is simpler, it will be easier to collect urine samples from a large group of

individuals. Moreover, biomarkers of LSDs are often more elevated in urine than in blood

(Meikle et al, 2004; Mills et al, 2005).

Urine samples are collected in high quantities but the transportation process can easily

alter the lipid content due to deterioration. For this purpose, the use of filter papers to support

and carry dried urine samples, is a more viable approach for sample collection (Auray-Blais et

al, 2007). In addition, filter paper samples can be sent by mail, facilitate the storage and do

not require pre-HPTLC preparation. To analyse filter paper urine samples by HPTLC it is

necessary to increase the method sensitivity. Te results obtained with the optimised HPTLC

method were similar to those of the original method in identification of FD patients that excrete

Gb3. This identification was possible by analysing urine samples contained in two filter papers,

which is a great progress, as filter paper samples are usually analysed by mass spectrometry

(Auray-Blais et al, 2006; Auray-Blais et al, 2008; Chien et al, 2011).



[50]

An efficient extraction procedure would extract all the lipid content from the filter paper

samples. Several factors are implicated in extraction efficiency, namely centrifugation,

sonication, type and duration of agitation and type of organic solvents. However, for filter paper

samples, centrifugation is not the best approach as it retains part of the extract resulting in a

decrease of the GSL band density.

The stored GSLs can be extracted with non-polar solvents, however, the membrane

bound GSLs are more polar, requiring polar solvents capable of breaking the hydrogen bonds

and electrostatic forces.

Diethyl ether and chloroform are both moderately polar solvents used for lipid

extraction, but diethyl ether is not a very good alternative since it forms peroxides on storage,

causing the risk of explosions.

The alcohols ethanol and methanol are the most polar solvents used for lipid

extraction. Methanol is extremely toxic to the operator, but unlike ethanol it is not contaminated

with aldehydes in storage and when combined with chloroform forms a general lipid extraction

solution.

GSLs may be attached to the cell membrane in amounts ranging from less than 5% (in

erythrocytes) to more than 20% of membrane lipids (in myelin of neurons) (Schnaar et al,

2009). Hence, the release of GSLs from the cell membrane requires a larger quantity of

methanol. The use of the conventional 1:2 (v/v) CHCl3/MeOH ratio seem to be the best

approach to extract neutral GSLs from filter paper urine samples, which together with

sonication and increase in agitation time, improved the extraction method, and as a

consequence, the efficiency of HPTLC.

As lipid extraction is a common procedure in lipid analysis techniques, this extraction

method can also be applied to mass spectrometry analysis of filter paper urine samples.

The used material can contaminate the lipid extracts with mineral oils, greases,

plasticizers from plastics, and detergents. Specifically plastics must be avoided because they

can cause the appearance of unknown spots in TLC plates. On the other hand, biological

samples also have to be purified from their own impurities, such as pigments, lipophilic amino

acids, polypeptides and hydrophobic proteins.



[51]

These substances can be removed with a NaCl/MeOH (v/v) solution in the

fractionation process and the exclusion of the polypropylene material from fractionation was

proved to further prevent the interference of contaminants in the HPTLC plate.

The compression of the silica particles in the C18 column and their ability to interact

with GSLs are also of most importance. The solutions used for GSL partition and vacuum

velocity, can influence the capacity of the C18 column to retain GSLs. The partition of GSLs

retained by the column is provided by a polar solution comprising MeOH/H2O (v/v), which

elutes gangliosides, and a less polar solution composed of chloroform/methanol (v/v), to elute

the neutral GSLs. The elution should be performed with the volume of solvent that allows the

best recovery of the lipids retained by the C18 column, which proved to be the volume

described in the original method.

The development distance is an important factor for GSL separation as it directly

influences the position of the bands. HPTLC has been described for the reduction in

development distance from the 10-15 cm necessary in TLC to 3-5 cm (Patel et al, 2011).

Although the development distance of 7 cm described in the original HPTLC method is over the

distance described in literature, the reduction of the development distance did not produce

satisfactory results. Further reduction of the development distance would be impracticable as it

would be more difficult to separate some substances, which may even merge due to similar

polarities.

GSL detection can be achieved with destructive reagents, such as spray of sulfuric acid

basis (p-anisaldehyde and orcinol), or nondestructive reagents, such as primuline spray.

Anisaldehyde is the derivatization agent with greater sensitivity for detection of GSLs on HPTLC.

The primuline spray was not explored since it requires UV light for lipid detection and its effect

is not permanent.

For the currently under development software, the establishment of the detection limit

is crucial, allowing the separation of GSL bands from the background without leading to false

positives. The optimised method has a Gb3 detection limit of 2.0 µg and guarantees that

samples with Gb3 bands stronger than that of 2.0 µg must be detected by the software.

The optimised HPTLC method revealed to be 13.6% more effective for Gb3 detection

than the conventional method, although some GSLs are lost in the procedure. However, the

efficiency of the method must tested by changing other conditions, such as the technician,



[52]

extraction temperature, fractionation velocity, TLC chamber humidity and temperature,

sonication temperature and humidity and temperature upon drying the filter paper.

The classification of an unknown sample as a potential FD patient (hemizygous and

heterozygous) or a non-FD patient, is the most important issue of a screening test. The

optimised HPTLC method identified Gb3 excretion in 60% of FD patients, of which, one patient

had borderline Gb3 levels between the FD and control groups.

It has been described that the genotype of FD hemizygous patients has some degree of

association with urinary Gb3 excretion levels (Auray-Blais et al, 2008; Vedder et al, 2007b).

The F113L was the most frequent mutation in this study, found among six patients.

Four of these patients presented Gb3 levels that would allow their identification in a blind

screening test. In relation to the other α-Gal A mutations represented in this study, the

available patients were not in sufficient number to allow the establishment of mutation-related

Gb3 excretion profiles. On the other hand, R118C and C94S patients present urinary profiles

according to literature.

All of the studied mutations are missense mutations and for that reason, no correlation

can be made between the urinary Gb3 and the type of mutation.

Analysing the differences between the GSL profile of samples collected before and

during the ERT, the improved HPTLC method proved to be suitable to follow up FD patients

under treatment. The new screening method identified high levels of Gb3 before the ERT that

decreased dramatically after three months.

The effect of ERT on Gb3 excretion in urine is important as ERT can reverse part of the

symptoms and decrease Gb3 excretion. On the other hand, the production of antibodies

against the enzyme may decrease the enzyme activity and as a consequence cause the

increase of Gb3 accumulation and excretion (Ohashi et al, 2007).

CHAPTER VI

6. CONCLUSIONS AND FUTURE PERSPECTIVES


Chapter VI – Conclusions and Future Perspectives

[54]

Several screening programs have been conducted in different high-risk or mass

screening populations. Most of these screening programs use mass spectrometry or mutational

analysis supported by determination of α-Gal A activity. However, these methods are very

expensive and time-consuming, requiring qualified technicians or complex and expensive

equipment.

The developed screening method offers an easy sample shipping method without

degradation of its constituents. Furthermore, it is suitable for a mass screening and ERT follow

up program as it can discriminate the affected individuals with high-risk of organ damage

caused by α-Gal A substrate accumulation and indirectly evaluate the therapy effect.

This method is easier to perform, its interpretation is clear and economically suitable to

use in a large population.

Future studies must contemplate the analysis of filter paper urine samples from a large

population of apparently healthy individuals and FD patients in order to refine the automatic

screening software. Filter paper urine samples of FD patients must be collected after the

establishment of protocols with their physicians and hospitals.


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8. APPENDIX I


Appendix I

[72]

Table III: Characteristics of control subjects. In the conventional method a 20 mL liquid urine sample is used. The filter

paper collection method refers to a 2 filter paper urine sample, with approximatelly 2 mL of urine per filter paper. M- Meles; F –

Females.

Control sample Collection Method Gender Age

1 Conventional F 26

2 Conventional M 20

3 Conventional M 62

4 Conventional F 23

5 Conventional M 33

6 Conventional M 49

7 Conventional M 28

8 Filter Paper M 51

9 Filter Paper M 32

10 Filter Paper F 28


12 Filter Paper M 30




















Appendix I

[73]


Appendix I

[74]

Figure 10: Filter paper kit instruction sheet for urine samples.

9. APPENDIX II


Appendix II

[76]

Table VIII: Densitometry of Gb3 bands from empty filter papers subjected to different extraction agitation

procedures. The Gb3 pixels were calculated by subtracting of background pixels to the total pixels of the Gb3 area. Gb3

values are expressed as mean ± standard deviation (SD). The variability of the analysis was calculated as relative SD (%RSD).

Lane Gb3

Mean ± SD (pixels)

%RSD

24 h 1610.1 ± 109.1 6.8

3x 2 h 1198.7 ± 505.5 42.2

5 min 1347.0 ± 2.5 0.2

Table XIII: Densitometry of Gb3 and SM bands of FD patients and control subjects. The Gb3 and SM pixels were

measured separately and the presented values are obtained by subtracting the background pixels to the total pixels of the Gb3

and SM area, respectively. Values are expressed as mean ± SD. The variability of the analysis was calculated as %RSD. n.d. –

non-detected.

Control Subjects

SM (pixels)

Gb3 (pixels)

Patients SM

(pixels) Gb3

(pixels)

C8 176.2 n.d. P6 400.1 n.d.

C9 53.4 n.d. P7 446.0 154.7

C10 121.9 n.d. P3 75.4 403.5

C11 41.7 n.d. P4 42.8 822.4

C12 33.4 n.d. P5 58.4 n.d.

C13 125.4 n.d. P2 84.7 372.9

C14 360.2 n.d. P8 19.9 n.d.

C15 134.6 n.d. P1 43.4 512.3

C16 250.9 n.d. P9 31.5 124.5

C17 469.4 n.d. P10 61.3 n.d.

C18 83.3 n.d. Mean ± SD 126.4 ± 157.9 398.4 ± 256.1

C19 71.6 n.d. % RSD 125.0 64.3

C20 26.4 n.d.

C21 401.4 176.4

C22 194.6 n.d.

C23 245.5 n.d.

C24 48.4 n.d.

C25 155.4 n.d.

C26 138.6 n.d.

C27 35.6 n.d.

C28 246.2 88.6

C29 n.d. n.d.

Mean ± SD 162.6 ± 127.1 132.5 ± 62.0

% RSD 78.2 46.8

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