Folate, vitamin B12, and

homocysteine

Charles H. Halsted, MD
Professor of Internal Medicine
April 2, 2004
Macrocytosis in peripheral blood,
folate or B12 deficiency
Megaloblastic bone marrow in
folate or B12 deficiency
Structure of folate in diet
 Sources of folate in the diet
Fortified cereal grains (1.4 mg folic acid /kg
grain)
Veggies: beans, lettuce, cabbage, spinach
Orange juice
Beans (various)
Organ meats: liver, kidney
Dietary Folate Equivalents (DFE)
of folate in food
Rationale: Food folate is 50% available
compared to 85% availability for folic acid,
so food folate is 50/85 = 0.6x available as
folic acid
Calculation: DFE (mg) = mg folate in a
given food product x 0.6 + mg supplemental
folic acid
 DRIs for folate (as DFEs)
Infants: 65-80 mg/d
Children (pre-pubertal): 150-300 mg/d
Adolescents: 400 mg/d
Adults: 400 mg/d
Pregnancy: 600 mg/d
Lactation: 500 mg/d
Folate homeostasis
 Folate deficiency: clinical settings
Dietary inadequacy and/or imbalance
Fast food and other imbalanced diet
Elderly malnutrition
Poverty
Intestinal malabsorption: celiac disease
Alcoholism: poor diet, absorption, liver
storage in cirrhosis
Other drugs: anticonvulsants, anti-inflammatories,
anti-folates all interfere with folate absorption or
metabolism
 Consequences of folate
deficiency

Megaloblastic anemia (DNA metabolism)

Neural tube and other congenital birth
defects
Hyperhomocysteinemia
Megaloblastic bone marrow in
folate or B12 deficiency
Folate metabolism and DNA synthesis
DNA

dTMP
Dietary folates

dUMP DHF

methionine

THF methionine synthase

ser
vitamin B6 vitamin B12
gly

5,10-MTHF homocysteine
MTHFR
5-MTHF

Dietary folates
 Neural Tube Defects

NTDs result from a disruption in neural tube

closure
spina bifida and anencephaly most common
occur during early pregnancy: neural tube closure
complete by post-conception day 28
Caused by folate deficiency and/or MTHFR
677TT (20%)
Most common birth defect in North America
2,500 US or 6/10,000 births per year
Mexican Americans: 13/10,000 births
Neural tube defect risk vs maternal red cell
folate levels, Dublin, Ireland 1980s
 MTHFR at the crossroads
DNA

dTMP
Dietary folates

dUMP DHF

methionine

THF methionine synthase

ser
vitamin B6 vitamin B12
gly

5,10-MTHF homocysteine
MTHFR
5-MTHF

Dietary folates
MTHFR variants associated with neural
tube defects and elevated homocysteine
C677T polymorphism
37% CC, 51% CT, 12% TT Hispanics >
Caucasians > African-Americans)
TT thermoliability of enzyme
Homocysteine elevation in TT ~1.5X
normal
Level is influenced by folate status, i.e.
high folate can overcome TT deficit
A1298C polymorphism: compound
heterozygotes
Folic Acid & Prevention of Neural Tube
Defects

Prevention:
400mg folic acid for women of child-bearing
age
all pregnant women take prenatal
supplements (0.8-1mg)
BUT must be provided before conception!
Development of dietary folate
deficiency
Laboratory diagnosis of folate deficiency

Test Abnormal value Interpretation

Serum folate < 5 ng/ml (11 nmol/l) Early sign, labile
Macrocytosis Early sign*
Red cell folate <160 ng/ml (350nmol/l) Reflects tissue stores
Plasma Hcy > 10 mMol/l) (varies) Metabolite*
Megaloblastosis Tissue deficiency*

* Non-specific since same finding occurs in B12 deficiency

Homocysteine level in diagnosis
DNA

dTMP
Dietary folates

dUMP DHF

methionine

THF methionine synthase

ser
vitamin B6 vitamin B12
gly

5,10-MTHF homocysteine
MTHFR
5-MTHF

Dietary folates
Diagnosis by homocysteine level
is sensitive but non specific
Vitamin B12 (cobalamin)
 Food sources and requirements for B12

Sources: meat, fish, poultry, milk products,

fortified cereals, (no vegetable source)
Requirements (mg/d):
Children 1.2
Adolescents 1.8
Adults 2.4
Pregnancy 2.6
Lactation 2.8
 Clinical features of B12 deficiency
Abnormal DNA synthesis and cell turnover
Anemia: increased MCV, macrocytosis, megaloblastic
bone marrow
Neurological abnormalities
White matter in brain; loss of cognitive fxn, ataxia
Subacute combined degeneration of spinal cord; loss of
position and vibratory sense
Elevated homocysteine associated with:
Cognitive defects of aging, ? Alzheimer disease
Enhanced vascular disease risk
Megaloblastic bone marrow in
B12 deficiency anemia
 White matter (myelin) degeneration in
brain of patient with B12 deficiency

Periventricular region Posterior column spinal cord

Vitamin B12 dependent reactions
Vitamin B12 absorption
Ileal enterocyte transport of vitamin B12
 Etiologies of B12 deficiency
Strict vegetarians Dietary
Elderly (15-20%) Lack of gastric acid
Use of antacid agent Lack of gastric acid
Pernicious anemia Lack of intrinsic factor (IF)
Gastric bypass Lack of acid and IF
Ileal disease/surgery Loss of absorbing surface
Immerslund/Grasbeck Loss of ileal receptor
Dietary causes of B12 deficiency
Essentially restricted to vegans who do not
use vitamin B12 supplements
high prevalence in India (>60%) and in Indian
immigrants who stick to dietary practices
Macrobiotic or other fad diets
Delayed onset since liver vitamin B12
stores are sufficient for 3-6 years
Liver stores and time for depletion
of folate vs vitamin B12
Gastric causes of B12 deficiency
Achlorhydria
aging (15% over aged 65)
Prolonged use of acid suppressant drugs
Pernicious anemia (2-3%)
Gastric surgery (mainly bypass)
Types of chronic gastritis

Type B Type A
Aging Pernicious anemia
Distribution of B12 levels by age
Prevalence of vitamin B12 deficiency in
those >65 yr is ~12%

Framingham study, 560 free living people ;

40% with low B12; 12% with high Hcy and
MMA

Denver outpatients: High Hcy and MMA found

in 12.5% of 150 patients
 Pernicious anemia
Described by Addison in 1849: fatigue,
macrocytic anemia in well nourished
individuals
Cause: antibodies to parietal cells and
intrinsic factor result in B12 malabsorption
Prevalence: 1.9 % of 729 elderly had PA
(2.7% women, 1.4% men; 4.3% in Black women )
Often associated with other antibodies, e.g. anti-
thyroglobulin
Gastric bypass with roux-y loop
Estimated Number of Bariatric Operations Performed in the United States, 1992-2003

Steinbrook, R. N Engl J Med 2004;350:1075-1079

A 76 yr old woman with macrocytic anemia
and neuropathy d/t ileal surgery
 Diagnosis of vitamin B12
deficiency

Low serum vitamin B12

Elevated MMA and Hcy (more sensitive)
Low TC II (?most sensitive)
Diagnosis by Hcy and MMA
 Vitamin B12 deficiency: Rx
Pernicious anemia, stasis, or ileal
abnormality: B12 injections (1000
mg/month) or high oral B12 (1 mg/d)
Gastric atrophy, partial gastrectomy or
bypass: oral B12 (350 mg/d).
Prevention by food fortification: may be
mandated in future (and makes sense)
 Folic acid treatment can cure
megaloblastic anemia while increasing
untilization of B12, thereby
masking B12 deficiency and
worsening its neurologic effects
 B12 and folate interaction

DNA

dTMP
Dietary folates

dUMP DHF

methionine

THF methionine synthase

ser
vitamin B12
gly

5,10-MTHF homocysteine
MTHFR
5-MTHF

Dietary folates
Hyperhomocysteinemia (hHcy)
Elevation in serum total Hcy >10 mmol/l
Etiologies
folate, B12, or B6 deficiencies,
polymorphisms in methionine cycle enzymes,
esp CBS and MTHFR
Effects: cardiovascular, (?) neural tube
defects, dementia in aging
Uncertain mechanism of cellular injury
 Methionine Cycle
DNA

S-adenosylmethionine Methyl donor

(SAM) -DNA
dTMP -phospholipids
folates
MAT -neurotransmitters

dUMP DHF

methionine S-adenosylhomocysteine
(SAH)
THF methionine synthase
ser
vitamin B6 vitamin B12
gly

5,10-MTHF homocysteine
MTHFR cystathionine -synthase
5-MTHF vitamin B6

cystathionine
folates
cysteine glutathione
Genetics of Hyperhomocysteinemia

Cystathionine -synthase (CBS)

Classified as pyridoxine-responsive or
non-responsive
Cause of congenital homocysteinuria
with early vascular disease; death
usually in 20s
Methylenetetrahydrofolate reductase
(MTHFR): neural tube defects,
cardiovascular, possibly cognitive, protection
against colon cancer
Odds ratio for CVD with
MTHFR 677TT
 Lifetime effects on homocysteine

Gender: Men > women

Age over 65 yr
Smoking
Coffee and/or alcohol in excess
Decreased renal function per serum
creatinine
Elevated homocysteine in elderly
Carotid stenosis and Hcy
Odds ratios: Hcy > 5 mmol/l increase vs
vascular disease
 RR for CVD; European
multicenter case vs control
Hcy and survival in CAD
 Folic acid fortification of U.S. food supply

Goal: to reduce serum homocysteine levels in at risk

individuals
Practice: 140 mg folic acid per 100 g grain (approx 300
mg/d DFE) since 1998
Effect: incidence of low folate reduced from 22 to
1.7%; hcy > 13 mMol/L reduced from 19 to 9.8%
Incidence of neural tube defect reduced by 20%, no
data on CVD risk