Anemia Biochem-Folate/B12, Iron, HMP shunt

Nutritional anemias are classified to three groups:

Microcytic (small red blood cell = RBC) defined by MCV smaller than 80 fentliters
-def in iron (major), copper, pyridoxine

Normocytic (normal size RBC) defined by MCV from 80-100
-protein-calorie malnutrition (kwashiorkor)

Macrocytic (large RBC) defined by MCV more than 100
-def in vitamin B12, folate (more common)

Megaloblastic anemia

folate and B12 essential for nucleotide synthesis

deficiency->megaloblastic

makes cell larger (S) phase takes longer time, stem cells

oval and larger in mature RBC

Folate metabolism

Dihydrofolate (FH2) + DHFR (dihydrofolate reductase)-->Tetrahydrofolate (FH4) (one carbon unit) (active form)

FH4->nucleotide biosynth or AA metab or B12-CH3 Meth metab

folic acid->convert to active form in two step rxn above. requires 2 NADPH (from Niacin/B3) and (DHFR). Each step adds 2 hydrogens to folate

FH4 pathways

Nucleotide synthesis
-de novo purine syn
-thymidine syn

AA metab
-serine synthesis (Gly->Ser or N-meythl Gly)

Meth metab
-homocysteine (hc) -> methionine

Folate components

absorbed as polyglutamate
needs to be reduced using NADPH (from niacin/B3) (uses dihydrofolate reductase)

3 parts of folate
1. ring
2. PABA
3. glutamate

dihydrofolate reductase is the target of methotrexate, inhibits folic acid

Folate function

transports one carbon units
used in DNA, AA and meth metab

Forms of FH4

FH4

FH4-C

-Formyl (N10-formyl FH4)->de novo purine, -CHO

-Methylene (N5, N10-methylene-FH4)->Thymidine (dTMP), Serine, -CH2-

-Methyl (N5-methyl-FH4)->B12-CH3, Methionine, -CH3

N10-formyl FH4 (Formyl = CHO) is used in:

De novo purine synthesis

N5, N10-methylene-FH4 (Methylene = CH2) is used in:

Thymidine (dTMP) synthesis

Serine synthesis (from glycine)

N5-methyl-FH4 (Methyl = CH3) is used in:

Methionine synthesis (from Homocysteine) via B12-CH3

The methylene form of folate can be converted into N5-methyl-FH4 via the enzymatic activity of _________________________.

Methylenetetrahydrofolate reductase (MTHFR)

Folic acid/B12-CH3 Methionine Metabolism

Meth->SAM->SAH->homocys

homocys is toxic->Meth (requires B12 and folic acid)

homocys (alt path)->cystathrionine->cysteine (dep on B6)

when hc goes up try to push to cys but has neg feedback (inhib)

will keep circling between meth, but hc will still build up

high hc is nemia in blood, uria in the urine

initially give folic acid, B12 and B6 for treatment

if B6 responsive or not, binds to enzyme to make cysteine. sometimes enzyme mutates and not binds

severe form of high hc

Hyperhomocysteinemia

Severe form (homocystinuria) is a rare (enzyme dep)

-CBS def, two types->B6 responsive and non-responsive (not based on activity, based on mutation site, bind or not)

less severe form is more common (5-7% total pop) (nutritional or genetic)

-most common is MTHFR def

-Folate/B12/B6 def

-Folate/B12 pathway def in other genes (MTR, MTRR, MMADHC)

Hyperhomocysteinemia lab conditions

Lab diagnosis:
-normal (5-15)
-moderate (15-20)
-intermediate (30-100)
-severe (>100)

normal value always given, if 10x going to be severe
in between is moderate or intermed
severe always genetic problem
less severe induced or dietary problem

Methyl-trap hypothesis

B12 deficiency blocks the reaction from FH4-CH3 (N5-methyl-FH4) to FH4

Folate is "trapped" as a form of FH4-CH3

This results in appearance of folate deficiency (but actually a B12 deficiency)

increases homocysteine

Biosynthesis of thymidine (dTMP)

thymidine synthase enzyme
-5-FU
-if knock out 5FU inhib both dTMP and FH4 pathway

MTX-inhibitor of FH2-FH4

MTX (methotrexate)

-used as therapy for rheumatoid arthritis

-side effects-inhib enzyme dihydrofolate reductase

-reduce immune cells, and rxns

also used in chemotherapy (inhibits cancer)

inhibits proliferation of lymphocytes and other cells responsible for inflammation in the join

MTX side effects (how to decrease)

DHFR-makes active form FH4 (from FH2)
coupled with B12

MTX inhibits DHFR, blocks up and downstream
goal is to knock out DNA nucleotide biosynthesis
knocks out many others too
can provide alternative form of tetrahydrofolate
-from food or supplement, alternative form of folic acid
-5-meythl form

Nutrition: B12 produced by bacteria

stored for 2 years in the liver
meat, eggs, dairy products good sources
not salad/veggie
also made by bacteria

Key role of B12 in stomach

B12 binds to intrinsic factor (IF) in stomach

-IF comes from parietal cells (IF is glycoprotein)

Transcobalamin (TC) then binds to B12 and carries to liver

1st issue-issue with parietal cell, if doesn't make IF, can't absorb B12, leads to B12 deficiency

-lose parietal cells due to damage or removal by surgery

-common autoimmune problem, gastric arthritis

2nd issue-stomach cancer, remove part of stomach that has parietal cells

Pernicious anemia

B12 deficiency due to lack of intrinsic factor (NOT RELATED TO FOLATE)

Current understanding of pernicious anemia (PA) is an _____________________ in which autoantibodies prevent the formation of the vitamin B12-IF complex, which in turn dramatically decreases vitamin B12 absorption.

autoimmune condition

The followings result in severe B12 deficiencies (lack of intrinsic factor) in the order of (from most common to rare):

Autoimmune gastritis (against parietal cells) (most common)

Surgery to remove stomach

Inherited disorders which prevent intrinsic factor production

Carbon (methyl) donors (how affect HC)

-folic acid->runs cycle to make HC->Meth, more or less good

-B12->same way as above

-SAM->if give SAM, increase SAM, increases HC/everything
makes condition worse, if HC worse, HCnuria/nemia worse

-Betaine (trimethylglycine)->alter pathway, HC->Meth
minor pathway, 3 methyl group makes Meth

1 carbon unit donors

Vitamin B12 deficiency

risk factor for cardiovascular disease

-(hyper)homocysteinemia

abnormal accumulation of FA in CNS

-leads to accumulation of methylmalonyl CoA (methylmalonic acidemia- MMA)

-MMA up specific to B12 (NOT FOLIC ACID)

-acidemia is the condition of high MMA (also called MMA)

-if MMA going up, pathway is blocked, FA not degrading, accumulate in body->typically in brain or CNS

Folate and B12 deficiencies

Folate and B12

-megaloblastic anemia

-high HC (total plasma)

--concen>13 mcmol/L abnormal, including cystathionine-B-synthase (CBS) deficiencies

--if concen 10x more then its genetic not nutrition

-neural tube defects (NTDs)

B12 deficiencies

-methylmalonic acidemia (MMA)

--measurement of methylmalonic acid (or methylmalonyl CoA) (MMA), distinguishes between B12 (cobalamin) and folate deficiencies.

--also can be caused by genetics, methylmalonyl CoA mutase deficiencies

if MMA HIGH->B12 ONLY

if MMA NORMAL->folate/B12 (probably both)

Cobalamin (Vitamin B12)

B12 has cobalt inside
original name cobalamin
different enzyme name

Free iron vs heme

anemia associated with iron
common health issue
easiest way to get iron is heme
not possible to absorb ionized free iron
can absorb some iron forms

Iron deficiency anemia

microcytic anemia

hypochromic-clear inside, can't make heme

MCV < 80

Iron distribution in a human

male, female distribution amount is the same
Hemoglobin heme component

ferritin-storage form iron
transferrin-small 1% form

Iron deficiency anemia causes

males-mostly absorption problem, inability to absorb iron, lack of iron in diet
females-blood loss, use of blood components, can be pregnancy

Iron is mainly absorbed in the duodenum and upper jejunum through either one of the following:

heme transporter --Heme and Fe2+ (using DMNT1 to to help absorb), heme best form for absorption

endosomes--Fe2+ (stored in ferritin)->once goes in as Fe2+ turns into Fe3+ (storage form) then when leaves converts back

FP1 transporter-takes Fe2+ out of ferritin, transports to TF (transferrin) to leave
-regulated by hepcidin (from liver), will block FP1/stop iron transport

TF-has two arms, can bind 2 irons

The major diet form of free iron Fe3+ needs to be converted to Fe2+ by:

Duodenal Cytochrome B (ferric reductase)
-gives an e-
-reduces so can use DMT1 transporter

Iron transport summary:

- Fe2+ (in the cells) is transported out from the cell through ferroportin (iron transporter).

- Fe2+ (outside of the cells) is immediately converted to Fe3+

- Fe3+ (outside of the cells) binds to transferrin

Iron absorption summary:

-Iron more readily absorbed in ferrous state (Fe2+)

-Most dietary iron is in ferric form (Fe3+)

- Fe3+ (a dietary form of free iron) is converted to Fe2+ by Duodenal Cytochrome B (= ferric reductase)

-gastric secretions: dissolve and form soluble complexes with ascorbic acid, reduction to the Fe2+ form

Inhibitors of iron absorbtion:

phytates, tannins, soil clay, laundry starch, iron overload, antacids

Competitors of iron absorption:

lead, cobalt, strontium, manganese, zinc

Facilitators of iron absorption:

ascorbate, citrate, amino acids, iron deficiency

Iron absorption, storage and transport

Iron Transport: Transferrin

Absorption: Free iron, heme

Iron Storage Ferritin

Iron Cycle

Transferrin takes iron from storage/ferritin (Fe2+ form) in the liver/instestine

TF1 binds to bone marrow to make heme
Heme packages RBC (life of 120 days)
Heme->recycled, free iron to make transferrin

Heme portion goes to heme degredation cycle->
RES (redticuloendothelial system) in macrophages
makes biliverdin->bilirubin (B)
B->BG->Bile

Iron storage

Ferritin stores the 3+ form
Fe2+ enters ferritin and is bonded with (OOH) to store as Fe3+
converted back to Fe2+ when leaves

Iron stores: sex susceptibility

Ferritin good marker of serum iron levels

normal male-goes up after about 20 years

normal female-goes up around 50 years due to menopause

females can maintain iron better than males, but can go down due to store depletion

Iron deficiency 3 phases:

-gradual, start

-middle-iron stores deplete, show deficiency, not anemia

-final-show phenotype, impact RBC, anemia

Iron absorption disease

Hemochromatosis, type 1

-HFE gene deficiency

-Too much iron absorbed from GI system

-preventable complications

-1/3 not show phenotype (reduced penetrance)

Rec for testing: phenotypic screening->genetic testing

HMP (hexose monophosphate shunt)

attached to upstream of glycolysis

2 functions:

NADPH (oxidative/irreversible)

DNA/RNA synthesis (non-oxidative/reversible)

G6P 2nd product after glucose->HMP, makes NADPH

other path back and forth with F6P, final destination is ribose 5P-sugar for nucleotide->makes purines and pyrimidines (can make gout worse)

G6PD deficiency-enzyme in first path, can't make NADPH (deficient in G6P dehydrogenase that makes 6-P gluconolactone from G6P). NADPH needed for GSH (antioxidant) leads to oxidative stress damage RBC

Von Gierke-can't make glucose from G6P, accumulates G6P->overproduce ribose 5-P, too many purines (hyperuricemia and gout)

Summary HMP shunt

Oxidative (irreversible)

-Glucose 6-P->>>2NADPH, Ribulose-5-P

-Enzyme: Glucose 6-P dehydrogenase (rate limiting)

Non-oxidative (reversible) (for nucleotides)

-ribulose-5-P->>>Ribose-5-P, G3P, F6P

-Enzyme: transketolases (cofactor vitamin B1) (thiamine)

thiamine impacts ribose5P, NOT NADPH

The five biochemical uses of NADPH are:

Scavenging hydrogen peroxide (H202) (protection from oxidative damage)

Phagocytic respiratory burst

Synthesis of nitric oxide (NO)

Reductive biosynthesis

Cytochrome P450 monooxygenase system

Uses of NADPH and GSH

Reduction of H2O2
H2O2 detoxed by GSH + enzyme GPx to make water
similar to catalase
GSH/GPx->cell membrane
GSH-made by glycine, cysteine, glutamate

GSH-attached form, can be recycled as GSSG
NADPH used to recycle
if NADPH knocked out->GSH stuck in inactive form (GSSG)
leads to accumulation H2O2, trouble in RBCs

Uses of NADPH and RBC

HMT shunt pathway generates NADPH to protect cell membrane lipids and proteins from oxidation, through regeneration of reduced glutatione

G6PD (glucose-6-phosphate dehydrogenase) deficiency

-blocks HMT shunt

-reduced supply of NADPH

-reduced GSH/increased oxidative stress (H2O2)

-causes hemolysis

Glucose 6-P dehydrogenase (G6PD) deficiency genetics

-X-linked, most common disease enzyme abnormality

-no clinical manifestations in most individ with def mutation

-hemolytic anemia with precipitating factors, including oxidants (oxidative stress)

--oxidative stress can cause the phenotype to express in individuals with mutation that didn't express before

-shows heinz bodies in RBCs

Precipitating factors in G6PH deficiency:

Ingestion fava beans (favism)

-all patients with favism have G6PD deficiency

-NOT all G6PD patients show favism

-creates oxidative stress

Use of an oxidant drug

Infection

Neonatal jaundice

NADPH for NOS

From Arginine to Citrulline

-nNOS (neuronal)

-eNOS (endothelial)

-iNOS (Inducible)->manny immune cells, macrophages, brain astroglia

NOS in OMM

NO:
-Endothelial cells (eNOS), which need NO for vascular smooth muscle relaxation (Ca2+ cGMP pathway)
-known as endothelium derived relaxation factor (EDRF)

OMT:
-can induce NO, leading to its analgesic, anti-inflam and relaxation effects

NAPDH for ROS respiratory burst

hydroxyl radical via iron-dependent pathway (fenton reaction)

peroxynitrite (or peroxynitrile) via iNOS pathway

hypolchlorus acid (HOCL) via myeloperoxidase-dependent pathway

Defects in phagocytic respiratory burst

Chronic granulomatous disease (CGD):

-immunodef, caused by defects in respiratory burst by immune cells

-formation of granulomas (nodular areas of inflammation)

-NADPH oxidase deficiences

--low (or no) superoxide (neutrophil function tests)

--can use hydrogen peroxide (H2O2) generated by invading organisms

-At high risk of infection by catalse-positive organisms (S. aureus and aspergillus)

Chronic granulomatous disease (CGD)

NADPH oxidase deficiency
-cant kill catalase positive bacteria (the bacteria will detox H2O2 and live and cause infection)
-can kill catalase negative bacteria (bacteria has hydrogen peroxide which is used to kill them)