Non Imaging In Vivo

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Non Imaging In Vivo

• Urine Test For Evaluation of
• B12 Absorption

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Vitamin B12

• B12 is essential for normal RBC production in
  bone marrow and normal liver cell metabolism.
• Vitamin B12 is not produced by plants or animals.
• It is actually produced by microorganisms found
  in soil and intestines and rumens (large first
  part of the stomach) of animals.
• Dietary B12 can naturally be found in animal
  foods including fish, milk and milk products,
  eggs, meat, and poultry.
• Fortified breakfast cereals are an excellent
  source of vitamin B12 and a particularly valuable
  source for vegetarians

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Uncommon to Be B12 Deficient

• Diets of most adult Americans provide recommended
  intakes of vitamin B12, but deficiency may still
  occur as a result of an inability to absorb B12
  from food.
• It can also occur in individuals with dietary
  patterns that exclude animal or fortified foods.
• As a general rule, most individuals who develop a
  vitamin B12 deficiency have an underlying stomach
  or intestinal disorder that limits the absorption
  of vitamin B12.
• Sometimes the only symptom of these intestinal
  disorders is anemia resulting from B12
  deficiency.

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Symptoms of B12 Deficiency

• Characteristic signs of B12 deficiency include
• Fatigue
• Weakness
• Nausea
• Constipation
• Flatulence (gas)
• Loss of appetite
• Weight loss

• Deficiency also can lead to neurological changes
  such as
• Numbness and tingling in the hands and feet
• Difficulty in maintaining balance
• Depression
• Confusion
• Poor memory
• Soreness of the mouth or tongue.

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B12The Short Story

• Vitamin B12, also called cyanocobalamin, is
  important to good health.
• It helps maintain healthy nerve cells and red
  blood cells, and is also needed to make DNA, the
  genetic material in all cells.
• Vitamin B12 is bound to the protein in food.
  Hydrochloric acid in the stomach releases B12
  from protein during digestion. Once released, B12
  combines with a substance called intrinsic factor
  (IF) before it is absorbed into the bloodstream.

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Absorption of B12

• For the body to absorb B12, it must be complexed
  with intrinsic factor (IF).
• IF is a protein secreted by parietal cells of the
  gastric fundus.
• The complex binds to receptors in terminal ileum
  in the presence of an alkaline pH and calcium,
  where B12 is actively transported across the
  mucosa.
• As B12 enters the portal vein of the liver, it
  binds to transcobalamin II, a transport protein.
• Then it is delivered to the liver.

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Absorption of B12 Continued

• Over the next 8-12 hours, portions of this B12
  reenters circulation binding to a larger
  transport protein, transcobalamin I.
• When the storage capacity of transcobalamin I is
  exceeded, B12 is excreted by the kidneys into the
  urine via glomerular filtration.
• As in, when we administer a flushing dose of B12,
  which we will discuss later.
• This is the basis of the test.

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Parietal cells of the gastric fundus secrete
Intrinsic Factor which binds to B12
IF/B12 complex binds to receptors in terminal
ileum and the B12 is actively transported across
mucosa
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B12 is Ingested
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B12 enters portal vein and binds to
Transcobalamin II and then enters the liver
All B12 not bound to transcobalamin I is excreted
out via kidneys and bladder
B12 re-enters circulation binding to
transcobalamin I
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Storage of B12

• B12 is primarily stored in the liver (a storage
  depot).
• Total body stores are high while daily excretion
  is low.
• This is why it takes 3-5 years to develop B12
  deficiency if dietary intake is halted or
  malabsorption occurs.
• Thus B12 deficiency due to diet is rare,
  occurring in strict vegetarians.

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B12 Deficiency Causes

• Inadequate Intake (rare)
• Malabsorption
• Absence of IF (pernicious anemia)
• Gastrectomy
• Excess HCI (Zollinger-Ellison Syndrome)
• Intestinal Absorption Problems
• Destruction, removal or invasion of ileal
  absorption sites
• Competition for B12 (tapeworm, bacterial
  overgrowth in small bowel lesions)

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B12 Deficiency Causes Continued

• Pancreatic disease
• Chronic Pancreatitis
• Cystic Fibrosis
• Causes failure of the pancreas to produce enzymes
  involved in breakdown of fats and their
  absorption from the intestine
• Medications
• p-aminosalicylic acid, Neomycin, colchicine,
  Prilosec, calcium-chelating agents
• Genetic abnormality in transport proteins

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B12 Deficiency Effects

• Megoblastic anemia
• Occurrence of large primitive red cell
• Thrombocytopenia
• A reduction in the number of platelets
• Leukopenia
• Reduction in the number of white blood cells
• Degeneration of the spinal cord
• Death
• Only if side effects cannot be reversed
• Note that hematological change is reversible,
  neurological may not be.

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Primary Reason for Absorption Test

• Pernicious anemia is a type of anemia caused by
  the bodys failure to absorb vitamin B12.
• Pernicious anemia is the most common cause of
  vitamin B12 deficiency.
• Pernicious anemia is characterized by the
  presence of anti-parietal cell and anti-intrinsic
  factor antibodies (50-80) leading to intrinsic
  factor deficiency and gastric mucosal atrophy.

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Indications for Schilling Test

• Low serum B12, with or without neurological or
  hematological symptoms.
• 2/3 of patients with low serum B12 have no signs
  or no symptoms
• Confirm the diagnosis of B12 malabsorption and
  determine the mechanism.
• Hematological changes with non-diagnostic serum
  tests.
• Detect patients at risk for B12 deficiency (e.g.
  post gastrectomy, ileal disease, family history
  of pernicious anemia).

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Isotopes Used in Schillings Test

• Cobalt 57
• 122 keV, half life of 270 days
• Cobalt 58
• 810 keV, half life of 71 days
• Cobalt 60
• 1170 keV, 1330 keV, half life 5.2 years
• Cobalt 57 is isotope of choice, why?

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Why Cobalt?

• The reason that cobalt is used as the
  radiopharmaceutical is because Vitamin B12
  (cyanocobalamin) has a non-radioactive form of
  cobalt as its central metal atom.
• Radioactive Cobalt can be substituted for the
  cold atom, producing a tagged form of B12.

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Pre-Test Concerns

• Confirm B12/Folate levels have been drawn and
  that the patient has a low B12.
• A normal B12 level virtually excludes B12
  deficiency.
• If absorption test is done prior to B12/Folate
  levels, labs checking for levels of B12 will not
  give true values. This is because B12 is
  administered in the Schilling Test.
• Folate deficiency can cause a megaloblastic
  anemia exactly the same as B12 deficiency except
  neurologic symptoms do not occur.

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More Pretest Concerns

• Ensure overnight fasting.
• The vitamin B12 from a meal can affect absorption
  (decrease it) leading to a false positive test.
• Confirm that no parenteral vitamin B12 has been
  given within the last three days.
• Enterohepatic circulation will compete with B12
  absorption from the ileum.

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Stage I B12 Absorption Test Technique

• Patient should be NPO for 12 hours
• Have patient void, administer 0.5 uCi of C0-57
  labeled Vitamin B12 in a 0.5 ug Vitamin B12
  capsule--orally.
• Up to 2 hours later, administer a flushing dose
  of 1,000 ug of cold Vitamin B12 intramuscularly
  or subcutaneously.
• This is to saturate transport proteins and
  ensures any radioactive B12 absorbed into the
  blood from the gut finds normal binding sites
  saturated and will be excreted via glomelular
  filtration into the urine sample.

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Stage I Technique Continued

• Collect and pool urine for 24 hours.
• 48 hours if there is renal impairment
• Maximum excretion is 8-12 hours after
  administration
• Co-57 labeled B12 absorbed thru GI tract will not
  be bound by saturated transport proteins and will
  thus be excreted in the urine.
• Measure volume for 24 hour urine collection

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Stage I Technique Continued

• Prepare standard
• Dilute 0.5 ml of Cobaltous Chloride Co-57
  provided with kit, with 3.5 ml of water.
• Standard solution contains the equivalent of 1
  of the total radioactivity in the oral dose.
• Pipette and count 4 ml aliquots of urine and dose
  standards for 10 minutes.
• Calculate percent administered dose excreted over
  (each) 24 hour period.

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Calculations

• Calculate the percent urine excretion of labeled
  B12 as follows
• (Avg.urine cpm Bkg cpm) x (Total urine vol.
  /counting volume) x 100
• --------------------------------------------------
  -------------------------------------
• (Std. cpm Bkg cpm) x Dilution
  Factor

• Dilution Factor is equal to 100, if the standard
  is a 1 of the dose.
• Example original concentration is 100 and the
  prepared standard is 1
• 100/1 100

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Results of Stage I

• Normal
• Greater than 10 of dose excreted in 24 hours
• Borderline
• 6-10 of dose excreted in 24 hours
• Abnormal
• Less than 6 of dose excreted in 24 hours
• Normally for pernicious anemia the result is 1-3

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Stage II Schilling Test

• If the Stage I test is abnormal, the exam is
  repeated by administering 0.5 ug of Co57 labeled
  with Vitamin B12 complexed to human intrinsic
  factor.

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Stage II Results

• Normal results indicate pernicious anemia
• No change in results indicates malabsorption
  instead of lack of IF
• Chronic B12 deficiency from PA can produce
  atrophy of ileal mucosa. This causes a decrease
  in intestinal absorption of B12.
• In these cases, there may be only a minor
  correction in Stage II.
• To diagnose this, repeat Stage II several
  weeks/months after institution of B12 therapy to
  allow mucosa to recover. (Stage III Absorption
  Test)

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Current Available MethodRubratope Diagnostic Kit

• Available through Squibb

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False Positive Results

• False positive results may occur in patients with
  diminished renal function or obstruction.
• In patients with extremely poor renal function,
  3-day collection should be performed.
• When the patient has multiple containers, the
  following should be done
• Percent excreted in second 24 hour sample should
  be added to first.
• If combined excretion is in the normal range,
  test is interpreted as normal.

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False Positive Results

• False Positive results can occur if a portion of
  the urine volume was lost.
• To verify all urine was collected
• check urine creatinine level
• Should be greater than 15 mg/kg/day.
• compare differences in volume between 24 hour and
  48 hour collections.

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False Positive Results

• Megoblastic anemia secondary to folate deficiency
• Veganism
• Third Trimester Pregnancy
• Contraceptives and anti-convulescents
• Multiple myeloma
• Radioactivity present in the urine prior to exam.

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False Positives

• The following drugs, can result in malabsorption
  of vitamin B12
• Most antibiotics, methotextrate, pyrimethamine,
  colchicine, para-aminosalicyclic acid, or
  excessive alcohol intake for longer than two
  weeks.

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False Normal Results

• Recent parental Vitamin B12
• Nitrous oxide inhalation
• Severe liver disease
• Chronic granulocytic leukemia
• Elderly patient
• H2 Blockers and iron deficiency anemia which lead
  to decreased gastric pH
• Fecal contamination

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Other Methods for Determining B12 Deficiency

• Instead of urine, an absorption test can be done
  by obtaining a stool sample 72 hours post isotope
  ingestion and count sample in a well counter.
  This expresses B12 not absorbed. Defective
  absorption is the problem if more than 70 of
  isotope is excreted fecally.

• There is also a plasma Schilling Test. 8-10
  hours after oral dose, draw 20 ml of blood and
  centrifuge. Draw plasma off of blood sample and
  count along with standard. Normal is .25 - 2.5.
  This test is good due to the fact it is very
  little patient dependant.

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An In-depth Review
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Deficiency leads to production of abnormal, large
red cells

• Vitamin B12 is a precursor of DNA synthesis. Lack
  of B12 impairs DNA synthesis within a cell, but,
  RNA and protein synthesis are unaffected.
• This results in dissociation between nuclear and
  cytoplasmic maturation, producing cells which
  have enlarged mature cytoplasm and immature
  nucleus (megaloblastosis).
• These findings are most prominent in cells with
  rapid turnovers--blood and GI tract. Thus, why
  B12 deficiency can lead to hematological
  changes--megaloblastic anemia or megaloblastic
  changes in the GI tract.
• If these megaloblastic changes occur in the
  terminal ileum, vitamin B12 absorption is
  inhibited.

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B12 Deficiency Causes Neurological Changes

• Vitamin B12 is also required for myelin
  metabolism therefore deficiency can cause
  neurological symptoms, classically involving the
  posterior columns and peripheral nerves leading
  to loss of position and vibratory sensation as
  well as degeneration of the spinal cord.

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Absorption Overview

• Ingested B12 is released from protein by
  digestive enzymes (gastric acid and pepsin).
• B12 binds to R protein in the stomach.
• R protein is found in gastric, biliary and
  salivary secretions.
• Pancreatic enzymes degrade B12-R and facilitate
  binding of B12 to Intrinsic factor, which occurs
  in the presence of an alkaline pH.
• B12-IF is absorbed by the terminal ileum.
• B12 enters serum bound to Transcobalamin-I and
  Transcobalamin-II.

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Conclusion

• Diagnosing B12 deficiency is imperative for
  patients long term recovery.
• The Rubratope Kit is easy, cost efficient, and
  give a direct evaluation of bodys ability to
  absorb B12.
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