83
Table 4.17 Trend of Age at Diagnosis in CKD of Participants
CKD Stage Age at diagnosis < 45 Yrs Age at diagnosis > 45 Yrs Odds Ratio
1 15 7 1.00
2 10 20 4.29
3 7 12 3.67
4 3 4 2.86
5 3 9 6.43
Abbreviation: CKD, chronic kidney disease Chi Square for trend 4.387
84
Table 4.18 Summary of Group Analysis of CKD Stage and mean values of Haematologic Variables of Participants
Variable Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
P-Value
ANOVA Bartlett's
Kruskal-Wallis ESR (mm in 1st hour) 28.64 28.64 46.69 56 72.27 0.027 0.019 0.012
Hb (g/dl) 11.85 12.18 11.77 7.65 7.57 0.001 0.943 0.001
Platelet (×109/l) 280.55 247.05 236.31 306.83 275.55 0.491 0.119 0.869
TWBC (×109/l) 6.53 7.28 5.03 7.72 7.35 0.130 0.001 0.018
Retic Count (%) 3.05 2.76 3.13 4.37 3.96 0.909 0.394 0.397
Serum Ferritin (µg/ml) 81.16 53.48 54.54 64.83 88.64 0.068 0.001 0.108
sTfR (ng/ml) 15.64 14.43 42.79 67.42 13.91 0.128 0.001 0.924
sEpo (IU/L) 8.59 6.26 29.54 38.83 5.32 0.114 0.001 0.511
Age at Diagnosis (Yrs) 37.96 48.64 43.93 43.83 52.46 0.054 0.111 0.059
4.5.10 Means analyses to assess significant variability of both the basic and special haematologic parameters among the participants.
Results: In the study group, there appears to be variation in the means of the haematologic parameters in each stage of CKD with a noticeable trend in ESR and Hb where it was noticed that with worsening stage of CKD (Stage 1 to 5 CKD) the ESR rises while Hb reduces. Statistical significance was found in those with a trend (Kruskal-Wallis 0.012 and 0.001 respectively) and in TWBC (Kruskal-Wallis 0.018). The rest of the variables even though appear with variability, they are not showing any trend, are disorganized and not statistically significant. Hence the variability noted might have just occurred by chance.
85 CHAPTER FIVE 5.0 Discussion
In this study it was observed that females predominate (54.9%) which contrasted with the finding of Shittu OA et al. in Ilorin North Central Nigeria, Ulasi II et al. in South East Nigeria, Abdu A. et al in Kano North West Nigeria and Iseki K in Okinawa Japan Asia.11, 133, 134, 135 Major risk factors for developing CKD, diabetes mellitus and hypertension are noted to be higher among the female participants, accounting for the number that came to the hospital. Further to that, the average body weight of females when compared to their male counterparts in the study showed that they have a lower average body weight and this has been associated with increased risk in CKD and end-stage renal disease (ESRD). 135
The study showed that most of the participants are unemployed 54 (59.34%), with females comprising 95% of the unemployed, hence giving a reason why there were more female patients that frequented the hospital. Additionally, of the 38 participants who had too frequent hospital visit 21 are females. This is because they are unemployed and are dependant on their spouses and relations for help which likely may make them present late to the hospital when the disease is advanced, warranting frequent close monitoring.
Mean age of participants (47±14.77 Years) is higher than that reported by Shittu AO et al. in Ilorin Central Nigeria133 and Ulasi II et al. in South East Nigeria, 134 where they reported mean age of 41.00±16.4 and 43.7±13.2 years respectively. This could be due to the higher education and enlightenment in the Southern Nigeria compared to the North136 accounting for better health seeking behaviour and early hospital presentation.
The majority of the participants (59.3%) were diagnosed at the productive age of 45 years, and this is in agreement with the age reported among African-Americans6. There is an age-related loss in estimated glomerular filtration rate (eGFR) as one advances in age, 137 and also race/ethnicity have been noted to be predictors of CKD.101 The odds for developing CKD with age show an increase with a significant association existing between age at diagnosis and development of CKD (p value 0.036, [Table 4.17]).
86
More males when proportionally compared to females were diagnosed at age 45 years and above as observed from the study (26 out of 41 males, 28 out of 50 females). This could be explained by the fact that women are protected from developing CKD and ESRD especially during their reproductive years as reported by Iseki K in 2008135, which is in agreement with their earlier centre-wide study in Okinawa Japan138, where they noticed that the male gender is a risk factor for worsening CKD. Though the deterioration of kidney function is the same in both genders, females develop CKD later than the males135, most likely due to nitric oxide, a vasodilator which is higher in women, especially pre-menstrual women.137 The higher risk of developing CKD in the male gender may be due to; risk factors for CKD such as hypertension, diabetes mellitus, smoking and large volume of alcohol consumption that is higher in men than women.135
The mean weight of the participants in the study is 66.47±16.51Kg, which is higher than that reported by Chijioke A et al 2009139 in Ilorin North Central Nigeria among CKD patients, where they got 63.7±11.9Kg. Low body weight has been shown to increase risk of both CKD and ESRD135, though no significant difference is seen between the genders (Table 4.3) in this study.
Common causes of CKD amongst studied participants are Hypertension (50.54%), Hypertension/Diabetes Mellitus (DM) (19.78%), Diabetes mellitus (6.59%) and Hypertension/Left Ventricular Hypertrophy (LVH) (5.49%). Although this is in agreement with other studies, 11, 12, 55, 69, 72Hypertensive disorders are more common in this study.
The prevalence of anaemia was found to be 61.5%, using an Hb of 12.0g/dl which is the Hb cut off for assessment of cause of anaemia in CKD for both sexes140. This figure is lower compared with those reported by Ijoma C et al (77.5%) in Enugu South East Nigeria, Shittu AO et al (94%) in Ilorin North Central Nigeria and Amoako YA et al (86.7%) in Ghana,104, 133, 141 but is higher than reported by McCellan et al (47.7%) in Atlanta U.S.A. This could be attributed to level of education and enlightenment towards the need of seeking early medical care of people in South East and North Central Nigeria, and also in Ghana. Lower reports from U.S.A could be as a result of better quality of care and so risk factors for CKD are promptly managed. Also race and ethnicity are predictors of development of anaemia in CKD.97, 101
87
The male-female prevalence of anaemia in CKD, Hb <13.5g/dl for male and <12.0g/dl for female, showed the prevalence to be 80.49% and 62.00% respectively (See Table 4.8). This is in agreement with the “kidney early evaluation program” (KEEP), and national health and nutrition examination survey (NHANES) of 1999-2004, which found anaemia to be more likely in males than females with CKD97. But a study done in Saudi Arabia gave a prevalence of 50% for male and 72% for female.140 McCellan et al have shown that female gender is a predictor of anaemia in CKD101, and also Ijoma C et al affirmed that anaemia in CKD is worse in female104. This is partly attributed to the normal physiological adaptation to anaemia in women where they tend to have reduced oxygen affinity, which is secondary to higher levels of 2, 3-diphosphoglycerate.142 Therefore male will present to the hospital earlier than female due to symptomatic anaemia.
Level of anaemia among studied participants revealed a predominant mild anaemia (48.5%) which was seen more in female within the age group of 25-54 years. Anaemia is a consistent finding in CKD in Zaria especially in the females. Female sex has been shown to be a predictor of anaemia and its worsening in CKD,101, 104and because of the age-related deterioration of kidney function and the highly active period of development, hence the age group 25-54 years predominating.
The mean haemoglobin level observed in this study was (10.97±2.81g/dl), this is however below the cut-off level of females and males as given by National Kidney Foundation-Kidney Disease Outcome Quality Initiative (NKF-KDOQI) (12.0g/dl, females and 13.5g/dl, males) and the European Best Practices Guidelines (11.5g/dl, females and 13.5g/dl, males)12, 55. It has been shown that in CKD, there is an overall reduction in iron 44, and iron deficiency is established when the mean serum Ferritin level fall below 100ng/ml44, 82 and Red cell distribution width is above 14.50%143, 144 respectively. Relating the result of this study where the mean serum ferritin and RDW-CV are 70.57±46.43ng/ml and 16.29±3.70 respectively, with the established cut off, support the fact of an existing iron deficient state. Red cell distribution width (RDW) percentage has been noted to be an early marker of iron deficiency.75, 145
88
This study revealed that the CKD patients in Zaria, North West Nigeria, have low levels of Reticulocyte percentage count, serum ferritin, soluble Transferin Receptor, and an apparently normal serum Erythropoietin level (3.18±4.07%, 70.58±46.44µg/ml, 22.90±49.70ng/ml and 12.49±33.47IU/L respectively). This agrees with earlier reports which show that the cause of
‘Renal Anaemia’ is reduction in iron44, relative erythropoietin deficiency and erythropoietin resistance13, 56, 65, 70, 113 accounting for poor reticulocyte receptor response. Even though Shittu AO et al. in Ilorin North Central Nigeria133, Talwar VK, Gupta HL and Shashinarayan in New Delhi India72 reported a high reticulocyte count, no reason was given for the findings in both studies.
There is a general difficulty in interpreting serum erythropoietin level in CKD, 65 but a ‘relative erythropoietin’ deficiency and resistance in CKD exists, 13, 56, 65, 66, 70, 113, 146 with a low set point for erythropoietin production by the kidneys, blighting the normal erythroblast receptor response, 66, 113 hence manifesting with impaired erythropoietin response113. Other reasons for the low erythropoietin production and receptor response is the relationship existing between CKD and inflammation, which results in cytokine release and dysregulation blunting the effect of erythropoietin on the marrow.55
Soluble transferrin receptor in this study 22.90±49.70ng/ml is low, compared with the accepted values in CKD.80, 82, 123, 147 This reflects the amount of iron supplied to the blood and the number of precursors, by extension the erythropoietic activity of the erythroblast.44 The reason for the low level could be attributed to erythropoietin resistance. Though the study revealed a normal serum erythropoietin level (Mean serum erythropoietin level 12.49±33.47IU/L is above 25th Percentile) 13, yet the expected increased elaboration of soluble transferrin receptors could not occur. This is in keeping with the explanation that ‘Renal anaemia’ is related to inadequate endogenous erythropoietin response to anaemia in CKD.13, 56, 70, 113 Serum ferritin levels remain a reliable marker of bone marrow iron stores and one of the most reliable initial diagnostic tests for iron deficiency, with a cut off for absolute iron deficiency in CKD set at 100ng/ml82. In this study serum ferritin level is 70.58±46.44µg/ml. This is explained by the fact that ferritin being an acute phase reactant 78, its production may have been affected by the inflammatory milieu and the low soluble transferrin receptor response co-existing in the CKD patients
89
studied. For reliable interpretation of serum ferritin, soluble transferrin receptor, which is a negative acute phase reactant, is advised to be assayed.44, 82, 123
The pattern of anaemia in CKD is predominantly that of normocytic, normochromic picture.
Means of MCV, MCHC, MCH (MCV, 83.31±8.74fl; MCH, 28.34±3.12pg; MCHC, 34.11±4.93g/dl) are all within normal range, consistent with most studies.11, 20, 71, 133The reason for this is that in
‘renal anaemia’ there is erythropoietin resistance and deficiency, leading to a blighted response, 11, 20, 56, 113, which leads to functional iron deficiency44, hence a predominant picture of normocytic and normochromic red blood cells, “the anaemia of erythropoietin reduction or resistance”.
Normal haematological values were observed for White blood cells (WBC), Platelet and red cell indices. This was noted in most Nigerian studies11, 55, 57, 133, and an Indian study 72 which noted that though cytokines have effects on blood cell survival, normal count is still observed. Fukami A et al in Japan observed an increase in WBC level but could not establish any causal relationship to the level of eGFR67, while Arun S. et al in South India observed leucopenia73. Though in CKD uraemia is frequently observed, (its effect is on function of WBC) it does not affect WBC production (granulopoiesis).133
High erythrocyte sedimentation rate (ESR), 40.11±37.76mm in 1st hour was observed in the study. This could not be unconnected to the level of haemoglobin because ESR, as an acute phase reaction rises in CKD. This is in keeping with what Arun S. et al observed in South India,73 and Afshar R. et al in Iran where they observed the rise in 50% of CKD patients.20 This is in contrast to the Ilorin report of Shittu AO.133 Even though ESR is high in this study, no association was noted between serum ferritin and ESR, possibly signifying that ESR cannot be used as an index of acute phase activity of serum ferritin in CKD. It can thus be concluded that measurement of ESR in CKD has no clinical utility.85
The study revealed statistically and clinically significant variability of haemoglobin and ESR in the study participants. Variability of such is noted in anaemia of CKD111, which cuts across the stages of CKD109, 111, signifying differences of mean in each stage of the disease. For the
90
Haemoglobin level, there is a fall in Haemoglobin as the disease progresses from stage 1-5. This positive correlation is consistent with most studies11, 20, 65, 104, 113 . The odd for a patient remaining anaemic all through the progression of the disease is significant with a p-value of 0.001 (Table 4.16). This relationship is showing an important characteristic in deterioration of kidney function.
There is a positive correlation between the ESR and stage of CKD, which means that as the disease worsens the ESR rises, this is in agreement with Bathon J et al85 and Arun S et al73. However this does not show any significant impact to the progression of the disease except when inflammation is considered.148
The study could not show any significant variability in serum ferritin, soluble transferrin, serum erythropoietin, platelet, WBC and reticulocyte count, with stage of CKD , but it, however, revealed an inverse relationship existing between the progressions of kidney disease parameters (negative coefficient noted), while a positive direct relationship existing with soluble transferrin receptors. This is on the background that apart from serum erythropoietin which is observed to be within the 25th percentile, soluble transferrin receptor and serum ferritin are all below the cut off.
A weak negative correlation was observed in the study between haemoglobin and ESR. The results are in agreement with other reports 66, 85, 93, 113, but in contrast with the Ilorin report 133 which showed otherwise. The significance of this is the fact that the relationship is an inverse one, where-by as the anaemia worsens across the CKD stages the ESR increases, the import of which will give need to monitoring disease progression.
In the study the relationship between Haemoglobin and serum erythropoietin is inverse, though with a very weak positive correlation (r˄2=0.02). A negative (inverse) correlation has been observed by many researchers20, 65, 66, 85, 113. This is what is seen when there is inadequacy of erythropoietin production by the kidneys in CKD20, while in this study it is more of erythropoietin resistance (serum erythropoietin is within 25th percentile which is generally accepted as the cut off for relative erythropoietin deficiency).13 The inverse relationship existing
91
between haemoglobin and serum erythropoietin is abolished when creatinine clearance gets above 30-40ml/min.65, 113
92 Conclusion
Findings from this study revealed that the CKD patient in Zaria is iron deficient with low transferrin receptor response to erythropoietin, manifesting as normocytic, normochromic
‘renal anaemia’. There is erythropoietin resistance where its interaction with sTfR and Hb is strong to moderate with a significant variation in Hb in all stages of CKD. Other relationship and associations where they exist are weak.
93
Limitation of the study
1. Hospital-based studies, like this, hardly give a true representation of the population as a sizeable number of potential participants will be seen in other facilities around.
2. The use of estimated glomerular filtration rate (eGFR) instead of measured glomerular filtration rate (mGFR)
3. A larger prospective study would have been better to establish key haematologic indicators in disease progression. A “before and after” study i.e. an interventional study is better at giving a clearer picture on relationships and associations
94
Recommendations
1. Government should, as a matter of policy, includes non-communicable diseases (NCDs) as public health diseases of importance with adequate financial support.
2. Database of NCDs should be encouraged through public private partnership
95 6.0 References
1. Alebiosu CO, Ayodele OE. The global burden of chronic kidney disease and the way forward. Ethnicity & disease. 2005;15(3):418-423.
2. Schieppati A, Remuzzi G. Chronic renal diseases as a public health problem:
epidemiology, social, and economic implications. Kidney Int Suppl. 2005(98):S7-S10.
3. Bosan IB. Recommendations for early diagnosis of chronic kidney disease. Annals of African medicine. 2007;6(3):130-136.
4. NKF-KDOQI. Clinical Practice Guidelines for Anemia of Chronic Kidney Disease: Update 2000. Am J Kidney Dis : the official journal of the National Kidney Foundation.
2001;37(1):S182-S238.
5. Atkins RC. The changing patterns of chronic kidney disease: the need to develop strategies for prevention relevant to different regions and countries. Kidney Int Suppl.
2005(98):S83-85.
6. Ojo A. Addressing the global burden of chronic kidney disease through clinical and translational research. Trans Am Clin Climatol Assoc. 2014;125:229-43; discussion 43-46.
7. Annear NMP, Banerjee D, Joseph J, Harries TH, Rahman S, Eastwood JB. Prevalence of chronic kidney disease stages 3–5 among acute medical admissions: another
opportunity for screening. Q J Med. 2008;101(2):91-97.
8. Afolabi MO, Abioye-Kuteyi EA, Arogundade FA, Bello IS. Prevalence of Chronic Kidney Disease in a Nigerian Family Practice Population. SA Fam Pract. 2009;51(2):132-137.
9. Fishbane S, Pollack S, Feldman HI, Joffe MM. Iron Indices in Chronic Kidney Disease in the National Health and Nutritional Examination Survey 1988–2004. Clinical journal of the American Society of Nephrology : CJASN. 2009;4(1):57-61.
10. Khanam S, Begum N, Begum S, Hoque AE. Changes in Hematological Indices in Different Stages of Chronic Renal Failure. J Bangladesh Soc Physiol. 2007;2:38-41.
11. Abdu A, Arogundade FA, Adamu B, Dutse AI, Sanusi A, Sani MU, et al. Anaemia and its response to treatment with Recombinant Human Erythropoietin in Chronic Kidney Disease Patients. W Afr J Med. 2009;28(5):295-299.
96
12. Bhatta S, Aryal G, Kafle R. Anemia in chronic kidney disease patients in pre-dialysis and post-dialysis stages. Journal of Pathology of Nepal. 2011;1(1):26-29.
13. Artunc F, Risler T. Serum erythropoietin concentrations and responses to anaemia in patients with or without chronic kidney disease. Nephrology Dialysis Transplantation.
2007;22(10):2900-2908.
14. Ferrari P, Kulkarni H, Dheda S, Betti S, Harrison C, St. Pierre TG, et al. Serum Iron Markers Are Inadequate for Guiding Iron Repletion in Chronic Kidney Disease. Clinical journal of the American Society of Nephrology : CJASN. 2011;6(1):77-83.
15. Rossert J, Fouqueray B, Boffa JJ. Anemia management and the delay of chronic renal failure progression. Journal of the American Society of Nephrology : JASN. 2003;14(7 Suppl 2):S173-177.
16. Ayodele OE, Alebiosu CO. Burden of chronic kidney disease: an international perspective. Adv Chronic Kidney Dis. 2010;17(3):215-224.
17. White SL, Chadban S, Jan S, Chapman JR, Cass A. How can we achieve global equity in provision of renal replacement therapy? Bull World Health Organ. 2008;86(3):229-237.
18. Bello AK, Nwankwo E, El Nahas AM. Prevention of chronic kidney disease: A global challenge. Kidney Int. 2005;68(S98):S11-S17.
19. Ulasi, II, Ijoma CK. The enormity of chronic kidney disease in Nigeria: the situation in a teaching hospital in South-East Nigeria. Journal of tropical medicine. 2010;(2010):50-57.
20. Afshar R, Sanavi S, Salimi J, Ahmadzadeh M. Hematological profile of chronic kidney disease (CKD) patients in Iran, in pre-dialysis stages and after initiation of hemodialysis.
Saudi journal of kidney diseases and transplantation : an official publication of the Saudi Center for Organ Transplantation, Saudi Arabia. 2010;21(2):368-371.
21. Nigerian Association of Nephrology. News Report. Nigeria2008; Available from:
www.nanephrology.org.
22. Amir H. Erythropoietin Friend or Foe in Chronic Kidney Disease Anemia: An Analysis of Randomized Controlled Trials, Observational Studies and Meta-analyses. Bri J Med Pract. 2009;2(3):12-20.
97
23. Odubanjo MO, Okolo CA, Oluwasola AO, Arije A. End-stage renal disease in Nigeria: an overview of the epidemiology and the pathogenetic mechanisms. Saudi journal of kidney diseases and transplantation : an official publication of the Saudi Center for Organ Transplantation, Saudi Arabia. 2011;22(5):1064-1071.
24. NKF. CPR 1.2.: Evaluation of Anemia in CKD. Am J Kidney Dis : the official journal of the National Kidney Foundation. 2006;47:S28-S32.
25. Couser WG, Remuzzi G, Mendis S, Tonelli M. The contribution of chronic kidney disease to the global burden of major non-communicable diseases. Kidney Int.
2011;80(12):1258-1270.
26. Arogundade FA, Sanusi AA, Hassan MO, Akinsola A. The pattern, clinical characteristics and outcome of ESRD in Ile-Ife, Nigeria: is there a change in trend? African health sciences. 2011;11(4):594-601.
27. Arogundade FA, Barsoum RS. CKD Prevention in Sub-Saharan Africa: A Call for
Governmental, Nongovernmental, and Community Support. Am J Kidney Dis: the official journal of the National Kidney Foundation. 2008;51(3):515-523.
28. Coresh J, Astor BC, Greene T, Eknoyan G, Levey AS. Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third national health and nutrition examination survey. Am J Kidney Dis. 2003;41(1):1-12.
29. Tsubakihara Y, Nishi S, Akiba T, Hirakata H, Iseki K, Kubota M, et al. 2008 Japanese Society for Dialysis Therapy: Guidelines for Renal Anemia in Chronic Kidney Disease.
Therapeutic Apheresis and Dialysis. 2010;14(3):240-275.
30. Diagnostic automation Inc. Calabass C. Product Insert. Serum Ferritin. Diagnostic automation Inc.; 2008. p. 1-5.
31. Awobusuyi JO, Kukoyi OO, Ibrahim MA, Atiba M. Indices of kidney damage and cardiovascular disease risk factors in a semiurban community of iloye, South-west Nigeria. International journal of nephrology. 2011;2011(1):1-7.
32. Feest TG, Mistry CD, Grimes DS, Mallick NP. Incidence of advanced chronic renal failure and the need for end stage renal replacement treatment. BMJ. 1990;301(6757):897-900.
98
33. Iseki K, Kohagura K, Sakima A, Iseki C, Kinjo K, Ikemiya Y, et al. Changes in the demographics and prevalence of chronic kidney disease in Okinawa, Japan (1993 to 2003). Hypertension research : official journal of the Japanese Society of Hypertension.
2007;30(1):55-62.
34. Lin HH, Tsai CW, Lin PH, Cheng KF, Wu HD, Wang IK, et al. Survival analysis of pediatric dialysis patients in Taiwan. Nephrology. 2012;17(7):621-627.
35. McGeown MG. Prevalence of advanced renal failure in Northern Ireland. BMJ.
1990;301(6757):900-903.
36. Kurokawa K, Nangaku M, Saito A, Inagi R, Miyata T. Current Issues and Future
Perspectives of Chronic Renal Failure. Journal of the American Society of Nephrology : JASN. 2002;13(suppl 1):S3-S6.
37. Takamatsu N, Abe H, Tominaga T, Nakahara K, Ito Y, Okumoto Y, et al. Risk factors for chronic kidney disease in Japan: a community-based study. BMC Nephrol. 2009;(10):10-34.
38. University of North Carolina Kidney center. A patient's guide to Chronic Kidney Disease:
Available from: www.unckidneycenter.org/kidneyhealthlibrary/CKD.
39. Barsoum RS. End-stage renal disease in North Africa. Kidney Int Suppl. 2003(83):S111-114.
40. Stewart P. Physiology of the Kidney. Update in Anaesthesia. 1998(9):1-3.
41. Turgut B, Ilhan N, Uyar FY, Celiker U, Demir T, Koca SS. Serum Erythropoietin levels in Patients with Central Serous Chorioretinopathy. Eye and Brain. 2010;2010(2):77-81.
42. Snively CS, Gutierrez C. Chronic kidney disease: prevention and treatment of common complications. Am Fam Physician. 2004;70(10):1921-1928.
43. Zoccali C, Kramer A, Jager KJ. Chronic kidney disease and end-stage renal disease - a review produced to contribute to the report 'the status of health in the European union:
towards a healthier Europe'. NDT Plus. 2010;3(3):213-224.
44. Urrechaga E, Borque L, Escanero JF. Assessing Iron Status in Chronic Kidney Disease Patients: New Laboratory Parameters. In: Gӧoz M, editor. Chronic Kidney Disease: CC BY 3.0 licence; 2012. p. 225-250.
99
45. Ulf S. Nutritional Aspects of Traditional Grains. Sweden1993; 1-6]. Available from:
wwwnysv.sik.se/traditionalgrains/review/.../Svanberg.pdf.
46. Okeke EC, Eneobong HN, Uzuegbunam AO, Ozioko AO, Umeh SI, Kuhnlein H. Nutrient Composition of Traditional Foods and Their Contribution to Energy and Nutrient intakes of Children and Women in Rural Households in Igbo Culture Area. Pakistan Journal of Nutrition. 2009;8(4):304-312.
47. Kwenin WKJ, Wolli M, Dzomeku BM. Assessing the Nutritional Value of some Afriican indigenous green leafy vegetables in Ghana. Journal of Animal and Plant Sciences.
2011;10(2):1300-1305.
48. Habwe FO, Walingo MK, Abukutsa-Onyango MO, Oluoch MO. Iron content of the formulated East African indigenous vegetable recipes. African Journal of Food Science.
2009;3(12):393-397.
49. Hallberg L, Hulthén L. Perspectives on Iron Absorption. Blood Cells, Molecules, and Diseases. 2002;29(3):562-573.
50. Conrad ME, Umbreit JN. Pathways of Iron Absorption. Blood Cells, Molecules, and Diseases. 2002;29(3):336-355.
51. Andrews NC. Disorders of Iron Metabolism. New England Journal of Medicine : NEJM.
1999;341(26):1986-1995.
52. Steele TM, Frazer DM, Anderson GJ. Systemic Regulation of Intestinal Iron Absorption.
IUBMB Life. 2005;57(7):499-503.
53. Lynch S. Case studies: iron. The American Journal of Clinical Nutrition. 2011;94(2):673S-678S.
54. Hsu C-y, McCulloch CE, Curhan GC. Epidemiology of Anemia Associated with Chronic Renal Insufficiency among Adults in the United States: Results from the Third National Health and Nutrition Examination Survey. Journal of the American Society of Nephrology : JASN. 2002;13(2):504-510.
55. O'Mara NB. Anemia in Patients With Chronic Kidney Disease. Diabetes Spectrum.
2008;21(1):12-19.