Non Imaging In Vivo

1. Non Imaging In Vivo Urine Test For Evaluation of B12 Absorption

2. 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

3. 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.

4. 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. Symptoms of B12 Deficiency

5. B12—The 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.

6. 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.

7. 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.

8. 1 2 Parietal cells of the gastric fundus secrete Intrinsic Factor which binds to B12 3 IF/B12 complex binds to receptors in terminal ileum and the B12 is actively transported across mucosa B12 is Ingested 5 6 4 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

9. 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.

10. 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)

11. 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

12. 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.

13. Primary Reason for Absorption Test • Pernicious anemia is a type of anemia caused by the body’s 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.

14. 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).

15. Isotopes Used in Schilling’s 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?

16. 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.

17. 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.

18. 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.

19. 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.

20. 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

21. 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.

22. 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 Calculations

23. 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%

24. 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.

25. 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)

26. Current Available Method—Rubratope Diagnostic Kit • Available through Squibb

27. 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.

28. 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.

29. 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.

30. 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.

31. 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

32. 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. Other Methods for Determining B12 Deficiency

33. An In-depth Review…

34. 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.

35. 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.

36. 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.

37. Conclusion • Diagnosing B12 deficiency is imperative for patient’s long term recovery. • The Rubratope Kit is easy, cost efficient, and give a direct evaluation of body’s ability to absorb B12. Return to the Table of Content