Megoblastic Anemia

Question 1

what is seen on a blood smear in a person with megoblastic anemia?

Answer 1

• erythrocytes that are either
  
  • normochromatic
  • and either
    
    • normal, or
    • VERY LARGE & misshapen (often oval)
• PMNs with hyper-segmented nuclei

Question 2

what is the general pathophysiology of megoblastic anemia?

Answer 2

• impaired DNA synthesis → reduction of nuclear divisions during erythroblast maturation → abnormally large RBCs
  
  • DNA synthesis is impaired d/t a defect in the transfer of one carbon groups, which depends on the formation of a one carbon pool. an insufficient one carbon pool is usually d/t
    
    • folate deficiency
    • Vit B deficiency

Question 3

what are the general steps / molecules required for the for the formation of the the carbon pool?

Answer 3

• the one carbon pool consists of a tetrahydrofolate group (FH4) that carries a carbon unit in either a formyl, methylene, or methyl form.
  
  • FH4 comes from FH4 synthesis, which requires:
    
    • folate
    • dihydrofolate reductase
    • NADPH
  • the carbon unit is donated from either:
    
    • amino acids - glycine, serine, histidine
    • formaldehyde / formate

Question 4

describe the structure of folate.

Answer 4

overall: pteridine ring + PABA (p-aminobenzoic acid) + glutamate(s)

• two forms:
  
  • ingested form: has several glutamates
  • absorbed form: has single glutamate (mono-form)

Question 5

tetrahydrofolate synthesis

• requires what molecules?
• involves what steps?

Answer 5

• requires:
  
  • folate
  • DHFR (dihydrofolate reductase)
  • 2 NADPH
• steps:
  
  • ingestion of folate
  • removal of extra glutamates in intestine
  • absorption of mono-form (pteridine ring+ PABA + single glutamate)
  • conversion of folate (F) → FH4
    
    • F reduced to FH2 by DHFR and NADPH
    • FH2 reduced to FH4 by DHFR and NADPH

Question 6

what are the sources of one-carbon units that are carried by FH4 in the carbon pool?

Answer 6

• glycine
• serine
• histidine
• formaldehyde
• formate

Question 7

attachment of the one-carbon unit to FH4

• requires what molecules?
• involves what steps?
• yields what forms of transferrable carbon?

Answer 7

three forms of carbon unit in carbon pool: formyl, methylene, methyl. they are synthesized in the following steps:

• FH4 → N¹⁰-formyl-FH4: requires ATP
  
  • carbon attached in the form of a “formate” group to 10th nitrogen of FH4
• N¹⁰-formyl-FH4 → N⁵,N¹⁰-methylene-FH4: requires NADPH
• N⁵,N¹⁰-methylene-FH4 → N⁵-methyl-FH4: requires NADH

Question 8

generation of what FH4-cabon product is the “least” reversible? why?

Answer 8

• N⁵-methyl-FH4 (synthesized from N⁵,N¹⁰-methyelene-FH4 using NADH)
  
  • the “methyl” form of carbon is CH3, and is thus as reduced as possible.
  • thus, N5-methyl-FH4 must donate its methyl group or stay methylated)

Question 9

what cellular processes require donation from the one-carbon pool? which form form of FH4 serves as the donor in each case?

Answer 9

• purine biosynthesis - formyl form
• dTMP synthesis - methylene form
• SAM reactions - methyl form

Question 10

purine biosynthesis requires carbon donation from what form of FH4?

Answer 10

formyl form: N¹⁰-formyl-FH4

Question 11

SAM (S-adenosyl methionine) reactions require carbon donation from what form of FH4?

Answer 11

methyl form: N⁵-methyl-FH4

Question 12

dTMP synthesis

Answer 12

methylene form: N⁵,N¹⁰-methyl-FH4

Question 13

what are the roles of NADPH in one-carbon unit transfer?

Answer 13

1. generation of FH4: used 2x by DHFR to reduce folate → FH2 → FH4
2. generation of N5,N10-methylene-FH4 (used in dTMP synthesis) from N10-formyl-FH4

Question 14

describe the structure of Vitamin B12.

Answer 14

= corrin ring + long chain + 3rd group

• corrin ring
  
  • has cobalt in the center
  • resembles heme
• 3rd group is one of the following:
  
  • methyl
  • adenosyl
  • cyanyl forms

Question 15

outline the absorption of Vitamin B12

Answer 15

Vit B12 consumed in protein rich dietary sources, then moved into the blood by several steps:

• Vit B12-protein carrier complex enters stomach
• here, the protein carrier is denatured, after which Vit B12 immediately becomes bound by other carriers:
  
  • initially - bound by R-binders made by gastric mucosa
    
    • this is brief, as R-binders are quickly degraded by acid
  • next - bound by intrinsic factor made by parietal cells
• Vit-B-intrinsic factor complex travels all the way to the ileum, where it:
  
  • is absorbed into the blood through ileal enterocytes, then
  • attached to transcobalamin-II, which transports it to
    
    • liver for storage
    • other tissues for use

Question 16

what is the role of intrinsic factor in the absorption of Vit B12?

Answer 16

• made by parietal cells
• are attached to Vit B12 in the stomach (after R-binders are degraded) then transport it to the ileum

Question 17

what is the role of transcobalamin II in the absorption of Vit B12?

Answer 17

• receives Vit B12 from intrinsic factor once the complex has entered the blood, then transports Vit B12 to
  
  • liver for storage
  • tissues for use

Question 18

what issues could disrupt cellular processes dependent on Vitamin B12?

Answer 18

• inadequate consumption of Vit B12
• decreased Vit B12 absorption, due to
  
  • gastric surgery
    
    • dec # of parietal cells → dec intrinsic factor production
  • aging
    
    • decreases HCl production → inhibited denaturing of dietary protein carrier → less free Vit B12

Question 19

what cellular processes require Vit B12?

Answer 19

only two reactions (but they are major)

1. regeneration of SAM (S-adenysoylmethionine)
2. conversion of metabolic products into succinyl CoA for entry into TCA cycle

Question 20

what is the role of Vit B12 in regeneration of SAM?

Answer 20

• Vit B joins N5-methyl-THF to form methyl-cobalamin, a group which can donate the methyl group to homocysteine to produce methionine + THF
  
  • methionine → SAM → methylation of various molecules

Question 21

what is the role of Vit B12 in the conversion of metabolic products into succinyl CoA? why is this important?

Answer 21

• Vit B joins methymalonyl CoA - a by product of 1. ketogenic metabolism and 2. odd-chain FA metobolism - allowing its conversion to succinyl-CoA
  
  • thus, allowing its entry into the TCA cycle

Question 22

SAM

• what molecules / steps are necessary for the generation of SAM?
• what is this importance of the SAM pathway?

Answer 22

• SAM synthesis
  
  • requires
    
    • N5-methyl-FH4
    • Vit B12
  • steps
    
    • N5-methyl-FH4 bound by Vit B12
    • this enables N5-methyl-FH4 to donate its methyl group to homocysteine, forming → methionine + THF
      
      • THF: recycled into carbon pool for re-use
      • methionine: converted to S-adenosyl methionine (SAM) by ATP
• importance of SAM pathway:
  
  • SAM methylatation is required for the production of several molecules, such as:
    
    • EPI
    • creatinine
    • methylated nucleotides
    • phosphatidyl choline
    • melatonin
  • serves to regenerate THF

Question 23

what does homocysteine come from?

how is homocysteine metabolized?

Answer 23

• homocysteine comes from a SAM molecule that has donated its methyl group
• homocysteine then processed in one of two ways:
  
  • converted into methionine for regeneration of SAM. requires
    
    • N⁵-methyl-THF
    • Vit B12
  • converted ty cystathionine → cysteine. requires
    
    • Vit B6

Question 24

what leads to hyper-homocysteinemia? explain.

Answer 24

• accumulation of homocysteine, due to either
  
  • impaired conversion to methionine
    
    • folate deficiency
    • Vit B12 deficiency
  • impaired conversion to cystathionine
    
    • Vit B6 deficiency

Question 25

clinical presentation of hyper-homocysteinemia?

Answer 25

several neurological + CV effects

Question 26

what is the “alternative pathway to methionine”? what is required for this pathway?

Answer 26

* a pathway (in the liver) that can partially _mitigate hyper-homocysteinemia_ by converting homocysteine to methionine * requires: **choline** * choline → betaine * betaine donates a methyl group to homocysteine, restoring methionine

Question 27

what biochemical pathways can somewhat mitigate hyper-homocysteinemia due to Vit B12 deficiency? what is the caveat of each?

Answer 27

* conversion of homocysteine → cystathione * this works temporarily, eventually _cysteine accumulates_ & inhibits cystathione generation by neg feedback * conversion of homocysteine → methionine by betaine (choline) * this only pathway _only works in the liver._ it does NOT stop the neurological affects of hyper-homocysteinemia

Question 28

what is the clinical presentation of hyper-homocysteinemia?

Answer 28

several neurological + CV effects

Question 29

what is the methyl trap hypothesis?

Answer 29

the idea that Vit B12 deficiency - by slowing conversion of homocysteine to methionine, leads to accumulation of * homocysteine * Methyl-FH4 this leads to * neurological sx * impaired folate recycling → inhibition of processes that require one carbon pool (purine synthesis, dTMP synthesis, ect)

Question 30

explain the features of erythrocytes in megoblastic anemia

Answer 30

seen in both folate & Vit B12 deficiency: * **abnormally large RBCs**: * _impaired DNA synthesis_ → fewer divisions → larger RBCs * impaired DNA synthesis d/t insufficient FH₄ b/c of: * insufficient folate - no FH4 production * insufficient Vit B12 - no FH4 recycling * **normochromic RBCs** * _hemoglobin production normal_

Question 31

what clinical features would help dx folate-deficient megoblastic anemia (vs Vit B12 megoblastic anemia?)

Answer 31

* presentation * folate deficiency, during pregnancy, can also lead to several fetal defects: * neural tube defects * spina bifida * anencephaly * dx * increased FIGLU levels (histidine metabolite)

Question 32

what clinical features would help dx Vit B12-deficient megoblastic anemia (vs folate deficient megoblastic anemia?)

Answer 32

* presentation * **several neurological sx** (likely d/t hyper-homocysteine) * _paresthesia_ - symmetrical, in hands & feet * _spastic gate_ - d/t position sense deficits * _irritability_ * _vision / taste disturbances_ * diagnosis * **elevated methylmalonic acid**

Question 33

methotrexate * what is its role? * how does it work?

Answer 33

* used in tx of cancer * **inhibits dTMP synthesis** by interfering with _one-carbon transfer_: * **blocks DHFR** → no conversion of FH2 to FH4 → no N⁵,N¹⁰=methylene-FH4

Question 34

5-FU * what is its role? * how does it work?

Answer 34

used in the tx of cancer * used in the tx of cancer * inhibits synthesis of dTMP * by serving as an analog of uracil (F-dUMP) blocking dUMP → dTMP