Vitamin B12 and folate deficiencies can cause megaloblastic anemia. The document discusses the roles of vitamin B12 and folate in DNA synthesis and cell maturation. It outlines the pathways of vitamin B12 and folate metabolism and consequences of deficiencies. Diagnosis involves evaluating mean corpuscular volume, blood smear examination, and levels of vitamin B12, folate, methylmalonic acid and homocysteine. Treatment involves vitamin B12 and folate supplementation.

1. Tinjauan Pustaka Hematologi ASPEK LABORATORIS ANEMIA MEGALOBLASTIK dr. Sri Kartika Sari/dr. Arifoel Hajat, SpPK Rabu, 16 Desember 2009

2. Pendahuluan Gangguan sintesis DNA Anemia megaloblastik Sel megaloblastik Ketidaksinkronan maturasi inti 2

3. Penyebab anemia megaloblastik 3

4. Vitamin B₁₂ Biokimia vitamin B₁₂ Methylcobalamin terbanyak dalam serum Protein transport : transcobalamin (TC) TC I, II dan III 4

5. P TISSUE Protein source Vitamin B₁₂ Oral intake Celullar uptake TC receptor STOMACH Parietal cell Pancreatic protease OH-Cbl CN-Cbl IF-binding LIVER IF-Cbl Holo-TC II Cubilin TC II Intestinal uptake storage 5 Gbr. Absorbsi, penyimpanan dan transportasi Vitamin B₁₂

7. Thymidilate

8. Histidine

9. Methionine

10. PurineMethyltetrahydrofolate terbanyak di serum 6

11. Metabolisme folat DNA dATP dGTP dTTP dCTP dTDP DHF polyglutamate THF polyglutamate dTMP 5,10 methylene THF polyglutamate THF dUMP methionine homocysteine Membran sel Methyl THF Methyl THF plasma Folat dari makanan Usus halus 7

12. 8 Gbr. Konversi Methylmalonil-coA dan sintesis methionine

13. Defisiensi vitamin B₁₂ 9

14. Intake kurang Kebutuhan malabsorbsi Defisiensi folat Gangguan metabolik kehilangan 10

15. Gambaran klinis Anemia berjalan lambat, memburuk perlahan Inefektif eritropoiesis Kelainan epitel Gejala defisiensi vitamin B₁₂ : Neuropati progresif gangguan metilasi mielin Gejala pada ibu hamil defek tabung saraf pada janin Peningkatan homocysteine miokard infark, vascular disease, trombosis vena. Kemandulan 11

16. Pemeriksaan laboratorium MCV Hapusan Darah Tepi Vitamin B₁₂ serum Folat serum dan eritrosit Methylmalonic acid (MMA) dan homocysteine Antibodi antiparietal cell dan anti IF Tes Schilling 12

17. Mean Corpuscular Volume (MCV) MCV > 100 fL Penyebab makrositosis : Megaloblastik atau non megaloblastik MCV meningkat sebelum terjadi penurunan kadar Hb. Bila defisiensi vitamin B₁₂ bersamaan dengan defisiensi besi MCV normal MCV kurang spesifik 13

18. Oval makrosit kesan penyakit megaloblastik Stomatosit dengan makrosit alkoholism Hipersegmentasi sensitif dan spesifik untuk anemia megaloblastik Hapusan darah tepi 14

19. Vitamin B₁₂ serum Digunakan untuk diagnosis dan monitoring terapi defisiensi vitamin B₁₂ Batas bawah normal biasanya sekitar 148 pmol/L (200 pg/mL) Belum ada baku emas  sensitivitas dan spesifisitasnya bervariasi lebar. Metode pemeriksaan : automated non-isotopic procedure menggunakan chemiluminescence 15

20. Lanjutan vitamin B₁₂ serum.... Pada gagal ginjal vitamin B₁₂ serum  tinggi Defisiensi vitamin B₁₂ pada penderita penyakit mieloproliferatif, gangguan ginjal/liver  kadar vitamin B₁₂ bisa normal/tinggi Kolonisasi bakteri usus  tinggi palsu 16

21. Folat serum dan folat eritrosit Batas bawah normal folat serum : 6,8 nmol/L Kadar folat serum  dipengaruhi diet. Kadar folat eritrosit  lebih mencerminkan simpanan folat jaringan. Belum ada metode baku emas 17

22. Methylmalonic acid (MMA) dan homocysteine Defisiensi vitamin B₁₂  MMA dan homocysteine Defisiensi folat  homocysteine MMA dan homocysteine  sensitif untuk diagnosis defisiensi vitamin B₁₂ Hati-hati interpretasi peningkatan metabolit bila tanda-tanda defisiensi (-) Setelah 7-14 hari pemberian terapi  kadar metabolit menjadi normal 18

23. 19

24. Antibodi anti sel parietal dan anti Intrinsic Factor (IF) Antibodi anti sel parietal : Non spesifik Terdapat pada penderita autoimun, orang sehat Antibodi anti IF : Tidak sensitif, namun cukup spesifik, walaupun masih mungkin ada pada penderita Graves disease. 20

25. Tes Schilling Untuk konfirmasi gangguan absorbsi vitamin Terdiri dari 2 tahap : 21

32. Gastrectomy

34. Pendekatan diagnosis penderita dengan kelainan hematologi Vitamin B₁₂ < 74 pmol/L >221 pmol/L 74-221 pmol/L Folat Folat N Folat N Folat Folat Folat N Defisiensi vitamin B₁₂ Defisiensi vitamin B₁₂ dan folat Bukan Defisiensi vitamin B₁₂ dan folat Def. folat Defisiensi vitamin B₁₂ Tx. folat Indeterminate Gbr.7 Tes Schilling Penyebab lain Gbr.7 Tes Schilling 23

35. 24 Gbr. 7. Pendekatan penderita dengan vitamin B₁₂ < 74 pmol/L

36. Tabel 6. Evaluasi penderita dengan kelainan hematologi dan vitamin B₁₂ <74 pmol/L 25

37. Gbr. 8. Evaluasi penderita yang diduga defisiensi vitamin B₁₂ dengan CBC normal 26

38. Diagnosis defisiensi vitamin B₁₂ dan folat hal yang kompleks. Belum ada tes yang mudah sebagai baku emasnya Perlu integrasi hasil laboratorium, klinis, respon terapi 27

39. TERIMA KASIH 28

40. 29 Gambar 6. Prinsip Beckman ACCESS Immunoassay System untuk pengukuran Vitamin B12. Serum.5

41. 30 Treatment B12 can be supplemented in healthy subjects by oral pill; sublingual pill, liquid, or strip; intranasal spray; or by injection. B12 is available singly or in combination with other supplements. B12 supplements are available in forms including cyanocobalamin, hydroxocobalamin, methylcobalamin, and adenosylcobalamin (sometimes called "cobamamide" or "dibencozide"). Oral treatments involve giving 250 ug to 1 mg of B12 daily.[28] Vitamin B12 can be given as intramuscular injections of hydroxycobalamin, methylcobalamin, or cyanocobalamin. Body stores (in the liver) are refilled with half a dozen injections in the first couple of weeks and then maintenance with monthly to quarterly injections throughout the life of the patient. B12 has traditionally been given parenterally to ensure absorption. However, oral replacement is now an accepted route, as it has become increasingly appreciated that sufficient quantities of B12 are absorbed when large doses are given. This absorption does not rely on the presence of intrinsic factor or an intact ileum. Generally 1 to 2 mg daily is required as a large dose [3]. By contrast, the typical Western diet contains 5–7 µg of B12 (Food and Drug Administration (FDA) Daily Value [29]).

42. 31

43. 32

44. 33 Hematological findings The blood film can point towards vitamin deficiency: Decreased red blood cell (RBC) count and hemoglobin levels[citation needed] Increased mean corpuscular volume (MCV, >95 fl) and mean corpuscular hemoglobin (MCH) Normal mean corpuscular hemoglobin concentration (MCHC, 32-36 g/dL) The reticulocyte count is decreased due to destruction of fragile and abnormal megaloblastic erythroid precursor. The platelet count may be reduced.[citation needed] Neutrophil granulocytes may show multisegmented nuclei ("senile neutrophil"). This is thought to be due to decreased production and a compensatory prolonged lifespan for circulating neutrophils, which increase numbers of nuclear segments with age.[citation needed] Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs). Macrocytes (larger than normal RBCs) are present. Ovalocytes (oval-shaped RBCs) are present. Howell-Jolly bodies (chromosomal remnant) also present. Blood chemistries will also show: In increased lactic acid dehydrogenase (LDH) level. The isozyme is LDH-2 which is typical of the serum and hematopoetic cells. Increased homocysteine and methylmalonic acid in B12 deficiency Increased homocysteine in folat

46. 35

47. 36

48. 37

49. Consequences of impaired folate status or metabolism Metabolic Disruption Biochemical Markers Clinical Associations CancerCVDdemyelinationNTDs S-adenosylmethionine (MTHFR, MS, B12 deficiency) Thymidylate Purines (A,G) Hypomethylated DNA Elevated homocysteine Reduced methylation CancerNTDsAnemia Increased uracil in DNADecreased DNA synthesis & reduced cell division Decreased DNA synthesis & reduced cell division Anemia

50. 39

52. sel neuroendokrin : gastrin

53. 41 Vitamin B12 Analogues Vitamin B12 is a coenzyme: it is needed for enzymes to do their job of changing one molecule into another. As vitamins go, B12 is large. One part of its structure is known as the corrin nucleus, which holds an atom of cobalt. The corrin resembles the heme of hemoglobin which holds an atom of iron. Any molecule that contains a corrin nucleus is considered a corrinoid. The corrin plus other atoms make up the cobalamin part of B12. There are many different cobalamins and they are named after their attachments. For example, methylcobalamin is cobalamin with a methyl group (one carbon and three hydrogens) attached. All corrinoids (including all cobalamins) are considered B12 analogues. Many corrinoids, and possibly even some cobalamins, are not useable by human B12 enzymes. These are considered inactive B12 analogues.

54. Vitamin B12 Deficiency homocysteine and methylmalonyl CoA Increase in methylmalonyl CoA Increased enzyme activity in fatty acid synthesis Build up of odd fatty acids around peripheral nerves Increase in homocysteine Vascular/nervous problems

56. 33% is carbohydrate

57. Molecular weight = 125,000-150,000

58. Beta globulin

59. Contains more sialic acid than transcobalamin III

60. Carries ~80% of Vitamin B12 in blood

62. Alpha globulin

63. NOT a glycoprotein

64. Carries less than 25% of Vitamin B12 in blood

65. Vitamin B12 has half-life of under 1 ½ hours when bound to it

66. Encourages absorption in a number of tissues

67. Degenerates once B12 is released

68. B12 then recirculates

70. Excreted in bile

72. 47

73. 48

74. 49 Reference Values Folat serum : > or = 3.5 ug/L

75. 50

76. 51

77. 52

78. 53

79. 54

80. 55

81. 56

82. 57 Methylmalonic acid (MMA) is a dicarboxylic acid that is a C-methylated derivative of malonate. Pathology Increased methylmalonic acid levels may indicate a vitamin B12 deficiency. However, it is sensitive without being specific. MMA is elevated in 90-98% of patients with B12 deficiency. This test may be overly sensitive, as 25-20% of patients over the age of 70 have elevated levels of MMA, but 25-33% of them do not have B12 deficiency. For this reason, MMA is not routinely recommended in the elderly. [1] An excess is associated with methylmalonicacidemia. MMA concentrations in blood are measured by Gas chromatographicMass spectrometry

83. 58 Folic acid and vitamin B12Large amounts of folic acid can mask the damaging effects of vitamin B12 deficiency by correcting the megaloblastic anemia caused by vitamin B12 deficiency [3,5] without correcting the neurological damage that also occurs [1,31]. Moreover, preliminary evidence suggests that high serum folate levels might not only mask vitamin B12 deficiency, but could also exacerbate the anemia and worsen the cognitive symptoms associated with vitamin B12 deficiency [6,11]. Permanent nerve damage can occur if vitamin B12 deficiency is not treated. For these reasons, folic acid intake from fortified food and supplements should not exceed 1,000 mcg daily in healthy individuals [5].

84. Diet Methyltetrahydrofolate Vitamin B12 Methionine Methyl B12 Homocysteine CH3 tetrahydrofolate Serine Dihydrofolate Purine and pyrimidine synthesis + B6 DNA Glycine Thymidylate Deoxyuridylate 5,10 methylenetetrahydrofolate Gambar 4. Jalur metabolik asam folat dan vitamin B12.2. 59

86. Lethargy

87. Weight loss

88. Weakness

89. Dementia

90. Leucopenia

91. Thrombocytopenia

92. Axonal degeneration

93. Demyelination

94. Urethral Sphincter problems

95. Depression

96. Alzheimer’s Disease

97. Increased liver weight

99. Homocysteine

100. MMA

101. Bilirubin excretion

102. LDH

103. Liver glycogen

104. Mitochondrial cristae in liver

105. Hepatic citrate synthase

106. Propionic acid

107. Succinate dehydrogenase

108. Cytochrome c activity

109. Propionyl CoA

110. Amino Acids

111. Cell metabolism

112. Protein synthesis

113. Fatty acid synthesis enzymes

114. ATP citrate lyase

115. Causes rise in Kreb’s cycle

116. Decrease

117. Transcobalamin II

119. 63 The present invention relates to a new method named the COBASORB test, which can be used for testing the cause of cobalamin malabsorption in humans. The COBASORB test contains three separate tests (first, second and third test) than can be performed separately, sequentially or in random order and number. The first test use non-radioactive cobalamin for ingestion, the second test uses non-radioactive cobalamin and recombinant intrinsic factor for ingestion and the third test uses recombinant haptocorrin saturated with cobalamin for ingestion. All three tests involve analysis of changes in the concentration of cobalamin saturated transcobalamin (holo-TC) and cobalamin saturated haptocorrin (holo-HC) in the blood. Also disclosed are fits suitable for use in these methods.

120. 64 Salivary haptocorrin, also known as the R-protein, binds strongly to Vitamin B12 (after it is released from food by gastric pepsin), stabilizing it and preventing its breakdown in the low-pH environment of the stomach. The complex is absorbed by ilealvilli into the blood. HC accounts for 10-40% of B12 serum level.