EXAMINATION OF SOME BIOCHEMICAL MARKERS OF NUTRITIONAL POSITION AMONG ADULT SICKLE CELL ANAEMIA PATIENTS IN STEADY STATE IN ZARIA
ABSTRACT
Sickle cell anaemia is a chronic haemolytic state which is characterized by hypercatabolism that could predispose to malnutrition. The low socio economic backgroundof our patients may worsen their health status. The study was aimed atassessing some biochemicalmarkers of nutritional status among adult sickle cell anaemia patients in steady state in Zaria. It is a crosssectional descriptive study which was conducted in the Departments of Chemical Pathology and Haematology, Ahmadu Bello University Teaching Hospital, Shika, Zaria, Kaduna State. Some biochemical markers of nutritional status of 60 adult SCA patients in steady state and 60 healthy non SCA controls were assessed. Mean (±SEM) serum vitamin D concentration was significantly lower in SCA patients 14.55±1.49 ug/L compared with controls 25.87.±4.29 ug/L (p<0.05). Mean (±SEM) serum calcium concentration was significantly lower in SCA patients 2.20 ±0.06 mmol/L compared with controls2.33±0.04 mmol/L (p<0.05).Mean (±SEM) serum albumin concentration was significantly lower in SCA patients 33.40±1.66 g/L compared with controls 40.13.±0.85 g/L (p<0.05).Mean (±SEM) serum inorganic phosphate concentration was significantly higher in SCA patients2.00.±0.13 mmol/L compared with controls 1.26±0.08 mmol/L (p<0.05).The mean (±SEM)BMI Kg/m2 was significantly lower in SCA patients 21.37±0.35 Kg/m2 compared with controls 23.27 Kg/m2 (p<0.05). The study also showedno correlations between BMI and the biochemical markers measured in both SCA patients and controls.
In conclusion, mean sera concentrations of vitamin D, calciumand albuminamong adult SCA patients in steady state were lowcompared with controls, so also BMI. The meanserum concentration of inorganic phosphate however, was significantly higher in SCA than in the control subjects. There wereno correlations between BMI and biochemical markers measured in thestudy population. Therefore SCA patients in steady state are more malnourished when compared to the controls.
TABLE OF CONTENT
Page
Title page………….…………………………………..……….………………..…….………. i
Declaration……………………………………………………………………….……… ii
Certification …………………………………………………………..…… ……………… iii
Acknowledgements………………………………………………………………………. iv
Abstract…………………………………………………………………………… ………v Table of contents…………………………………………………………… ………………vi
List oftables..…………………………………………………………………….………. x
List offigures……………………………………………………………………..…..…. xi
Abbreviations/Symbols used…………………………………………..……………….. xii
1.0 CHAPTER ONE: INTRODUCTION.…………………………………………. 1
1.1 Background…………………………………………………..……………………. 1
1.2 Statement of the problem…………………………………………………………. 2
1.3 Justification……………………………………………….………………………. 3
1.4 Aim and objectives…………………………………………………………..…… 3
1.5 Research question/Hypothesis…………………………………………………….. 4
2.0 CHAPTER TWO: LITERATURE REVIEW………………………………… 5
2.1 Sickle cell anaemia………………………………………………………………. 5
2.1.1 Historyof Sickle Cell anaemia….……………………………………………………… 5
2.1.2 Epidemiology of Sickle cell disease…………………………………………….. 5
2.1.3 Pathophysiology of Sickle cell anaemia………………………………………… 6
2.1.4 Clinical Features……………………………………………………………..…. 9
2.1.4.1 General Features………………….…………………………………………….. 9
2.1.4.2 Painful Crises…………………………………………………………………… 9
2.1.4.3 Infections…………………………………………………………..…………… 10
2.1.5 Effects on major organ systems…………`……………………………………… 10
2.1.5.1 The Respiratory System……………………………………………………… 10
2.1.5.2 The Cardiovascular system……………………………………………….…. 11
2.1.5.3 The liver and spleen………………………………………………………….. 11
2.1.5.4 The Musculoskeletal System……..………………………………….……… 11
2.1.5.5 The Nervous System…………………………………………………………. 12
1.5.6 The Genito-Urinary system…………………………………………………… 12
2.1.6 Sickle Cell anaemia and Nutrition…………………………………………… 13
2.1.6.1 SCA and Undernutrition…….………………………………………………. 14
2.1.6.2 Effect of Infection on nutrition in SCA………………………………. ..…. 16
2.1.6.3 Implications for growth and maturation abnormality in SCA…………… ……. 16
2.1.6.4 Nutritional intervention in management of SCA……………………..….. 17
2.1.7 Diagnosis of Sickle Cell Disease…………………………………………….. 18
2.1.7.1 Laboratory Diagnosis………………………………………………………… 18
2.1.7.1.1Haemoglobin sickling test……………………………………… ………….… 18
2.1.7.1.2Haemoglobin solubility test………………………………………………… 18
2.1.7.1.3Haemoglobin electrophoresis……………………………………………….. 19
2.1.7.1.4 Isoelectric focusing………………………………………………………..… 19
2.1.7.1.5 High Performance Liquid Chromatography……………………………….. 20
2.1.7.1.6DNA analysis………………………………………………………………… 20
2.1.8 Samples used in antenatal Diagnosis………………………………………… 21
2.1.8.1 Foetal blood sampling…………………………………………………….… 21
2.1.8.2 Chorionic villi or amniotic cell DNA………………………………………. 22
2.1.9 Treatment of sickle cell anaemia…………………………………………… 22
2.1.9.1 Drug treatments…………………………………………………….……… 22
2.1.9.1.1Hydroxyurea……………………………………………………………….. 23
2.1.9.2 Transfusion………………………………………………………………… 23
2.1.9.3 Bone Marrow or Stem Cell Transplantation………………………………… 24
2.2 Methods for assessing nutrition………………………………………………..… 25
2.2.1 Clinical observation and examination………………………………………………………. 25
2.2.2 Anthropometric measurement…………………………………………………. 26
2.2.3 Dietary history…………………………………………………………………… 26
2.2.4 Biochemical methods…………………………………………………………… 26
2.3 Vitamin D………………………………………………………………………. 28
2.3.1 Vitamin D metabolism………………………………………………………… 28
2.3.2 Mechanism of action………………………………………………………….. 29
2.3.3 Functions of vitamin D……………………………………………………….. 30
2.3.4 Vitamin D deficiency…………………………………………………………. 33
2.3.5 Risk factors for vitamin D deficiency………………………………………….. 35
2.3.6 Assessing vitamin D nutritional status in SCA……………………………… 39
2.3.6.1Vitamin D assay…………………………………………………………… 39
2.3.6.2 Extraction and Deproteinization………………………………………. …… 40
2.3.6.3 Column Chromatography……………………………………………………… 43
2.3.6.4Measurement of 25-Hydroxyvitamin D………………………… …………… 43
2.3.6.5Measurement of 1,25-Dihydroxyvitamin D……………………………… …….. 43
2.3.6.6Radioreceptor Assay…………………………….………………………………. 44
2.3.6.7Radioimmunoassay………………………………………….………………….. 44
2.3.6.8 Specimen Requirements……………………………………………………..… 44
2.4Albumin…………………………………………………………………………. 45
2.4.1 Synthesis of albumin…………………………………………………………… 46
2.4.2 Degradation of albumin…………………………………………..……………. 47
2.4.3Role of albumin in maintaining microvascular integrity………………….… 47
2.4.4 The prognostic value of serum albumin………………………………………… 48
3.0 CHAPTER THREE: MATERIALS AND METHODS…….……………….…. 50
3.1 Study area…………………………………………………………………….. 50
3.2 Study population and Design…………………………………………….. 50
3.2.1 Inclusion criteria……………………………………………………………. 51
3.2.2 Exclusion criteria……………………………………………………………. 51
3.2.3 Informed Consent…………………………………………………………. 51
3.2.4 Ethical consideration………………………………………………………… 51
3.2.5 Sample size determination..……………………………………….…………. 52
3.3 Sampling technique…………………………………………..………….…… 53
3.4 Blood collection and processing……………………………..……….……… 54
3.5 Chemicals……………………………………………………………….……. 54
3.6 Equipment…………………………………………………………………… 54
3.7 Quality control………………………………………………………..……… 54
3.8 Analytical methods………………………………………………………… 55
3.8.1 Measurement of serum Vitamin D………………………………………… 55
3.8.2 Measurement of serum calcium…………………………………………… 56
3.8.3 Measurement of serum phosphate………………………………………….. 57
3.8.4 Measurement of serum albumin………………………………………….. 58
3.9 Statistical analysis……………………………………………………………. 59
4.0 CHAPTERFOUR: RESULTS…………………………………………………… 60
5.0 CHAPTER FIVE: DISCUSSION……………………………………………… 66 6.0 CHAPTER SIX: CONCLUSION AND RECOMMENDATION…………… 69
6.1 CONCLUSION………………………………………………………………..… 69
6.2 RECOMMENDATIONS……………………………………………………….. 69
REFERENCES………………………………….…………………………………… 70
APPENDICES………………………………………………………………………… 92 LIST OF TABLES
Table Page
4.1 Anthropometric characteristics ofthe study population ………………..…. 61
4.2 Some biochemical markers of nutritional status in the study population….. 62
4.3 Correlationsbetween BMI and some markers of nutritional statusamong
adult SCA patientsin steady state…………………………………………. 63
4.4 Correlations between BMI and some markers of nutritional status in control…… 64 LIST OF FIGURES
FigurePage
2.1 Picture showing sickled red blood cells…………………………………..…………….…8
2.2 Diagram showing synthesis of vitamin D…………………………………………….…..41 2.3 Diagram showing calcium and phosphate regulation…………………………………….42 ABBREVIATIONS AND SYMBOLS
| AA | Atomic Absorption | |||
| AAS | Atomic Absorption spectroscopy | |||
| ABU | Ahmadu Bello University | |||
| ABUTH | Ahmadu Bello University Teaching Hospital | |||
| ACS | Acute chest syndrome | |||
| ALB | Albumin | |||
| ARDs | Acute Respiratory syndromes | |||
| ATP | Adenosine triphosphate | |||
| AVN | Avascular necrosis | |||
| BCG | Bromocresol Green | |||
| BMD | Bone mineral density | |||
| BMI | Body mass index | |||
| BMT | Bone marrow transplant | |||
| CA | Cellulose acetate | |||
| Ca2+ | Calcium ions | |||
| cAMP | Cyclic Adenosine monophosphate | |||
| CDC | Centre for disease control | |||
| cGMP | Cyclic Guanosine monophosphate | |||
| CLSI | Clinical Laboratory and Standards Institute | |||
| cm/s | centimetre per second | |||
| CPBA | competitive protein binding assay | |||
| CPC | CresolphthaleinComplexone | |||
| DBP | Diastolic blood pressure | |||
| DNA | Deoxynucleic acid | ||
| EDTA | Ethylinediaminetetraacetic acid | ||
| eg. | Example | ||
| etc. | etcetera | ||
| EIA | Enzyme imminoabsorbant | ||
| ELISA | Enzyme-linked ImmunoSorbent assay | ||
| et al | And others | ||
| FGF | Fibroblast growth factor | ||
| Fig. | Figure | ||
| Gc-globlin | Group-specific component globulin | ||
| GFR | Glomerular filtration rate | ||
| GIT | Gastrointestinal tract | ||
| Hb | Haemoglobin | ||
| HbA | Haemoglobin A | ||
| HbF | Haemoglobin F | ||
| HbPhiladelphia | Haemoglobin Philadelphia | ||
| HbS | Haemoglobin S | ||
| HbS Lep B | Haemoglobin S Lepore | ||
| HbS O Arab | Haemoglobin S O Arab | ||
| HbSC | Haemoglobin SC | ||
| HbSD | Haemoglobin SD | ||
| HbSD Punjab | Haemoglobin SD Punjab | ||
| HbSE | HaemoglobinSE | ||
| HbSS | Haemoglobin SS | ||
| HIV | Human immune deficiency virus | ||
| HPLC | High performance liquid chromatography | ||
| ICMA | Immunochemiluminescence assay | ||
ID-MS isotope dilution-mass spectrometry
IEF Isoelectric focusing
IL Illinois
Invitro Outside body
Invivo Inside body
ISEs Ion selective electron
IU International unit
KDa kilodalton
Kg kilogramme
Kg/m2 Kilogramme per metre square
Km Kilometre
LCMS liquid chromatography-mass spectrometry LDH Lactate dehydrogenasem mg/dl milligramme per deciltre
ml milliliter
MAPK mitogen-activated protein kinase
mmHg Millimetre of mercury mmol/L millimole per litre
mol mole
n Sample size
NADP nicotinamide-adenine dinucleotide phosphate ng/ml nanogramme per millilitre
nm nanometre nmol nanomole
NO Nitric oxide
NSAID Non steroidal anti-inflammatory drug
NW Normal weight
0c Degree Celsius
P Probability
PCR Polymerase chain reaction pH -log of hydrogen ion concentration
PO32+ Phosphate ions
PTH Parathyroid hormone
RANKL Receptor activator of nuclear factor-kBligand,
RBC Red blood cell
RCTs Randomized controlled trials
RFLP Restriction fragment length polymorphism
RIA Radioimmunoassay
RNA Ribonucleic acid
rpm revolution per minutes RRA Radioreceptor Assay
SBP Systolic blood pressure
SCA Sickle cell anaemia
SCD Sickle cell disease
SEM Standard error of mean
SNPs Single nucleotide polymorphisms
SPSS Statistical Package for the Social Science TAMV Time-average mean velocity u micro
ug/L microgramme per litre umol/L micromol per litre
UV Ultra violet
UVB Ultra violet B
VCAM -1 vascular cell adhesion molecule 1
| VD2 | Vitamin D 2 | |||
| VD3 | Vitamin D 3 | |||
| VDBP | Vitamin D binding protein | |||
| VDR | Vitamin D receptor | |||
| VTD | Vitamin D | |||
| VTDD | Vitamin D deficiency | |||
| WBC | White blood count | |||
| WHO | Whole Health Organization | |||
| α | Alpha | |||
| γ | Gamma | |||
| δ | Delta | |||
| α- | Alpha thalassaemia gene (minor) | |||
| α+ | Alpha thalassemia gene (major) | |||
| β | Beta | |||
| β- | Beta thalassemia gene (minor) | |||
| β+ | Beta thalassemia gene (major) | |||
| % | Percent | |||
| ± | Plus or minus | |||
| ≤ | Equal to or less than | |||
| ≥ | Equal to greater than | |||
| < | Less than | |||
| > | Greater than | |||
| ≈ | Approximately | |||
CHAPTER ONE
1.0 INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Sickle cell anemia (SCA) is a genetic disease that results from the substitution of valine for glutamic acid in the β-globin chain of the hemoglobin molecule (Pauling and Itano, 1949). The consequence of this amino acid substitution is the formation of hemoglobin S (HbS). Under low oxygen tension and/or conditions of acidosis. HbS precipitates and forms polymerized crystals called tactoids (hemoglobin polymers), which distort the red blood cells (Nelson and Cox, 2005; Ganong, 2003). The resulting sickle-shaped red cells lose their pliability and cannot navigate the small capillaries, become sticky, and adhere to the small veins, small arteries, and other blood vessels causing vaso-occlusion (Aster, 2005; Bunn and Forget, 1977). In addition, red blood cells homozygous for HbS (HbSS) are susceptible to premature destruction, with a red blood cell life span of 8–25 days as compared to 100–120 days for normal red blood cells (Solankiet al, 1988).
In the last few decades, studies have documented the presence of micro- and macronutrient deficiency among individuals with SCA and their possible association with immunologic, nutritional and growth abnormalities (Heymanet al.,1985; Gray et al., 1992; Serjeant et al., 2001). Patients with sickle cell anemia (SCA) have low bone mass compared to healthy subjects (Lalet al, 2006). Low bone mass in these patients is apparent even after adjusting for age, height, pubertal development, and lean body mass, suggesting that the deficits cannot be fully explained by short stature, delayed puberty, or altered body composition. Chronic haemolytic anemia and the resulting erythroblastic hyperplasia may contribute to bone demineralization in SCA (Serjeant and Serjeant, 2001). Furthermore, reduced physical activity, decreased circulating growth hormone, vitamin D deficiency and poor dietary intake of bone-forming nutrients are likely contributing factors (Buisonet al, 2004). Suboptimal peak bone mass acquisition in childhood may contribute to the development of osteoporosis in later life (Heanyet al, 2002).
Studies using direct measure of nutritional status (Enwonwu and Lu, 1991; Gray et al., 1992; Kennedy et al., 2001), indirect assessment of nutritional status (Henderson et al., 2005), and application of nutritional supplementation (Prasad and Cossack, 1984, Heymanet al., 1985), have established the association between SCA and the presence of nutritional deficiency among patients with the disease. These studies showed that although intake might be sufficient when measured against the recommended daily dietary allowance for age and sex, it is still insufficient for the individual with SCA due to the increased nutritional demand imposed by the disease. The result was the manifestation of malnutrition-like features (Prasad, 1997; Al-Saqladiet al, 2008; Hyacinth, et al, 2010).
1.2 STATEMENT OF THE PROBLEM
Nutritional deficiencies have not been given serious consideration in the assessment of SCA patients in this environment. It will be essential for physicians to adopt a nutritional approach as a part of the management modality for SCA in the light of the fact that more than two-thirds of the patients with SCA live in areas with low socioeconomic status and have little to no means of accessing the current methods of management. Therefore the present study is aimed at assessing some biochemical
markers of nutritional status in this group of patients.
1.3 JUSTIFICATION OF THE STUDY
The assessment of nutritional status in SCA patients could improve in the management of their conditions.
1.4 AIM AND OBJECTIVES OF THE STUDY
Aim
To assess some biochemical markers of nutritional status among adult SCA patients in steady state in Zaria.
Objectives
The objectives of this study were as follows:
- To determine serum levels of vitamin D, calcium, phosphate and albumin in adult SCA patients in steady state in Zaria and HbAA controls.
- To compare the serum levels of vitamin D, calcium, phosphate and albumin obtained from adult SCA patients in steady state and apparently healthy non SCA HbAA controls.
- To determine the anthropometric parameters of adult SCA patients in steady state and HbAA controls.
- To correlate the anthropometric parameters with serum levels of vitamin D, calcium, phosphate and albumin obtained from the study population.
1.5 RESEARCH HYPOTHESIS
H0: Sickle cell anaemia does not affect nutritional status (Null hypothesis).
H1: Sickle cell anaemia affects nutritional status (Alternate hypothesis).
EXAMINATION OF SOME BIOCHEMICAL MARKERS OF NUTRITIONAL POSITION AMONG ADULT SICKLE CELL ANAEMIA PATIENTS IN STEADY STATE IN ZARIA