Non Imaging In Vivo Urine Test For Evaluation of B 12 Absorption

Vitamin B 12 B 12 is essential for normal RBC production in bone marrow and normal liver cell metabolism. Vitamin B 12 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 B 12 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 B 12 and a particularly valuable source for vegetarians

Uncommon to Be B 12 Deficient Diets of most adult Americans provide recommended intakes of vitamin B 12, but deficiency may still occur as a result of an inability to absorb B 12 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 B 12 deficiency have an underlying stomach or intestinal disorder that limits the absorption of vitamin B 12. Sometimes the only symptom of these intestinal disorders is anemia resulting from B 12 deficiency.

Symptoms of B 12 Deficiency Characteristic signs of B 12 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.

B 12 —The Short Story Vitamin B 12, 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 B 12 is bound to the protein in food. Hydrochloric acid in the stomach releases B 12 from protein during digestion. Once released, B 12 combines with a substance called intrinsic factor (IF) before it is absorbed into the bloodstream.

Absorption of B 12 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 B 12 is actively transported across the mucosa. As B 12 enters the portal vein of the liver, it binds to transcobalamin II, a transport protein. Then it is delivered to the liver.

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

B 12 is Ingested Parietal cells of the gastric fundus secrete Intrinsic Factor which binds to B 12 IF/B 12 complex binds to receptors in terminal ileum and the B 12 is actively transported across mucosa B 12 enters portal vein and binds to Transcobalamin II and then enters the liver B 12 re-enters circulation binding to transcobalamin I All B 12 not bound to transcobalamin I is excreted out via kidneys and bladder

Storage of B 12 B 12 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 B 12 deficiency if dietary intake is halted or malabsorption occurs. Thus B 12 deficiency due to diet is rare, occurring in strict vegetarians.

B 12 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 B 12 (tapeworm, bacterial overgrowth in small bowel lesions)

B 12 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

B 12 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.

Primary Reason for Absorption Test Pernicious anemia is a type of anemia caused by the body’s failure to absorb vitamin B 12. Pernicious anemia is the most common cause of vitamin B 12 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.

Indications for Schilling Test Low serum B 12, with or without neurological or hematological symptoms. 2/3 of patients with low serum B 12 have no signs or no symptoms Confirm the diagnosis of B 12 malabsorption and determine the mechanism. Hematological changes with non-diagnostic serum tests. Detect patients at risk for B 12 deficiency (e.g. post gastrectomy, ileal disease, family history of pernicious anemia).

Isotopes Used in Schilling’s Test Cobalt keV, half life of 270 days Cobalt keV, half life of 71 days Cobalt keV, 1330 keV, half life 5.2 years Cobalt 57 is isotope of choice, why?

Why Cobalt? The reason that cobalt is used as the radiopharmaceutical is because Vitamin B 12 (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 B 12.

Pre-Test Concerns  Confirm B 12 /Folate levels have been drawn and that the patient has a low B 12. A normal B 12 level virtually excludes B 12 deficiency. If absorption test is done prior to B 12 /Folate levels, labs checking for levels of B 12 will not give true values. This is because B 12 is administered in the Schilling Test. Folate deficiency can cause a megaloblastic anemia exactly the same as B 12 deficiency except neurologic symptoms do not occur.

More Pretest Concerns –Ensure overnight fasting. The vitamin B 12 from a meal can affect absorption (decrease it) leading to a false positive test. –Confirm that no parenteral vitamin B 12 has been given within the last three days. Enterohepatic circulation will compete with B 12 absorption from the ileum.

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

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 B 12 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

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.

Calculations Calculate the percent urine excretion of labeled B 12 as follows: (Avg.urine cpm – Bkg cpm) x (Total urine vol. /counting volume) x (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

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%

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 B 12 complexed to human intrinsic factor.

Stage II Results Normal results indicate pernicious anemia No change in results indicates malabsorption instead of lack of IF Chronic B 12 deficiency from PA can produce atrophy of ileal mucosa. This causes a decrease in intestinal absorption of B 12. 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 B 12 therapy to allow mucosa to recover. (Stage III Absorption Test)

Current Available Method— Rubratope Diagnostic Kit Available through Squibb

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.

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.

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.

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.

False Normal Results Recent parental Vitamin B 12 Nitrous oxide inhalation Severe liver disease Chronic granulocytic leukemia Elderly patient H 2 Blockers and iron deficiency anemia which lead to decreased gastric pH Fecal contamination

Other Methods for Determining B 12 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 B 12 not absorbed. Defective absorption is the problem if more than 70% of isotope is excreted fecally. There is also a plasma Schilling Test hours after oral dose, draw 20 ml of blood and centrifuge. Draw plasma off of blood sample and count along with standard. Normal is %. This test is good due to the fact it is very little patient dependant.

An In-depth Review…

Deficiency leads to production of abnormal, large red cells Vitamin B 12 is a precursor of DNA synthesis. Lack of B 12 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 B 12 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 B 12 absorption is inhibited.

B 12 Deficiency Causes Neurological Changes Vitamin B 12 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.

Absorption Overview Ingested B 12 is released from protein by digestive enzymes (gastric acid and pepsin). B 12 binds to R protein in the stomach. R protein is found in gastric, biliary and salivary secretions. Pancreatic enzymes degrade B 12 -R and facilitate binding of B 12 to Intrinsic factor, which occurs in the presence of an alkaline pH. B 12 -IF is absorbed by the terminal ileum. B 12 enters serum bound to Transcobalamin-I and Transcobalamin-II.

Conclusion Diagnosing B 12 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 B 12. Return to the Table of ContentTable of Content