Your SlideShare is downloading. ×
0
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Anemia & vitamins
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

Anemia & vitamins

2,797

Published on

Vitamins and Anemia: …

Vitamins and Anemia:
Your body needs vitamins ( nutrients found in most foods) for many reasons, including producing healthy red blood cells. If your body is deficient in certain key vitamins, you can develop a type of anemia ( a condition in which your blood is low on healthy red blood cells ) called vitamin deficiency anemia.

Red blood cells carry oxygen from your lungs to all parts of your body. Without enough healthy red blood cells, your body can't get the oxygen it needs to feel energized. To produce red blood cells, your body needs iron and certain vitamins along with adequate protein and calorie intake.

Vitamin deficiency anemia can also lead to other health problems. Fortunately, you can usually correct vitamin deficiency anemia with supplements and dietary changes.

Published in: Education, Health & Medicine
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
2,797
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
126
Comments
0
Likes
1
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide
  • June 26, 2012 Conspicuous= آشکار،هویدا Speculative= قابل تعمق
  • June 26, 2012 Among the more obscure causes of megaloblastic anemia is the acronymic curiosity thiamine-responsive megaloblastic anemia (TRMA), subject of an article by Boros and colleagues (page 3556 ). The use of mass spectrometry in conjunction with stable isotope-labeling techniques has made it possible to unlock doors along previously inaccessible hallways of gene function analysis in the metabolomic maze. The door to TRMA was thus opened by Boros et al, who have pioneered the use of stable isotope-based dynamic metabolic profiling (SIDMAP) as a key to better understanding of changes in substrate flow as a basis for drug mechanisms and disease. Teaming up with the Boston group who first identified the loss of function mutation in the high-affinity, low-capacity thiamine transporter in TRMA, the authors have pinpointed the cause of disruption of nucleic acid synthesis that leads ultimately to premature apoptosis in this intriguing genetic disorder. Through tracking the stable 13 C-labeled glucose in fibroblasts from patients with TRMA, these authors concluded that the underlying lesion in this condition resides in the pentose cycle, specifically the transketolase enzyme, which requires thiamine pyro-phosphate as a cofactor. Through a consideration of the several interconnected pathways of glycolysis, the tricarboxylic acid cycle, and ribose synthesis, the authors defined substrate flux in TRMA and normal wild-type fibroblasts grown in both low- and high-thiamine medium. They concluded that defective high-affinity thiamine transport in TRMA leads to a critical reduction in de novo generation of ribose with consequent cell-cycle arrest that triggers precocious apoptosis. Their results clearly demonstrate a selective and time-dependent loss of ribose synthesis in TRMA patients that is most marked under thiamine-deprived culture conditions and is partially restored by thiamine supplementation, explaining the clinical responsiveness of TRMA patients to high doses of thiamine. Use of the powerful tools provided by SIDMAP and related techniques that use even more sensitive accelerator mass spectrometry with ultra-low-dose labeling techniques provides the promise to address, perhaps in vivo, similar unanswered questions involving the molecular basis for disease. Applying these methods to the study of the more common conditions that cause megaloblastic anemia, but that are still shrouded in mystery, could ultimately shed similar light on their mechanism. Ralph Green University of California at Davis References Wickremasinghe RG, Hoffbrand AV. Reduced rate of DNA replication fork movement in megaloblastic anemia. J Clin Invest. 1980;65: 26-36. [Medline] [Order article via Infotrieve] Goulian M, Bleile B, Tseng BY. The effect of methotrexate on levels of dUTP in animal cells. J Biol Chem. 1980;255: 10630-10637. [Abstract/ Free  Full Text] Koury M, Horne D. Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia. Proc Natl Acad Sci U S A. 1994;91: 4067-4071. [Abstract/ Free  Full Text]
  • June 26, 2012 Ameliorates = بهبود یافتن
  • June 26, 2012 Heme biosynethsis begins in the mitochondrion with the formation of 5-aminolevulinic acid. This molecule moves to the cytosol where a number of additional enzymatic transformations produce coproporphyrinogin III. The latter enters the mitochondrion where a final enzymatic conversion produces protophorphyrin IX. Ferrochelase inserts iron into the protophorin IX ring to produce heme.
  • June 26, 2012 Babinski reflex: Alternative Names     Reflex - Babinski's; Extensor plantar reflex Definition     Return to top Babinski's reflex occurs when the great toe flexes toward the top of the foot and the other toes fan out after the sole of the foot has been firmly stroked. This is normal in younger children, but abnormal after the age of 2. Considerations     Return to top Reflexes are specific, predictable, involuntary responses to a particular type of stimulation. Babinski's reflex is one of the infantile reflexes. It is normal in children under 2 years old, but it disappears as the child ages and the nervous system becomes more developed. In people more than 2 years old, the presence of a Babinski's reflex indicates damage to the nerve paths connecting the spinal cord and the brain (the corticospinal tract). Because this tract is right-sided and left-sided, a Babinski's reflex can occur on one side or on both sides. An abnormal Babinski's reflex can be temporary or permanent. Causes     Return to top Generalized tonic-clonic seizure (there may be a temporary Babinski's reflex for a short time after a seizure ) Amyotrophic lateral sclerosis Brain tumor (if it occurs in the corticospinal tract or the cerebellum) Familial periodic paralysis Friedreich's ataxia Head injury Hepatic encephalopathy Meningitis Multiple sclerosis Pernicious anemia Poliomyelitis (some forms) Rabies Spinal cord injury Spinal cord tumor Stroke Syringomyelia Tuberculosis (when it affects the spine) Home Care     Return to top Typically, a person (older than an infant) who has a Babinski's reflex will also have incoordination , weakness , and difficulty with muscle control. Safety is important to prevent the risk of injury. The person may need assistance with activity, and the environment should be kept free of hazards. When to Contact a Medical Professional     Return to top This finding is usually discovered by the health care provider, and the affected person usually is not aware of its presence. What to Expect at Your Office Visit     Return to top The medical history will be obtained and a physical examination performed. Medical history questions will be asked documenting this reflex in detail. The physical examination will probably include a complete nervous system (neurologic) examination. Diagnostic testing may include: MRI scan of the head or MRI scan of the spine Angiography of the head Somatosensory evoked potentials Lumbar puncture and analysis of the cerebrospinal fluid --------------------------------
  • June 26, 2012 Bone marrow aspiration: Alternative Names     Iliac crest tap; Sternal tap Definition     Bone marrow is the tissue that makes blood cells. It is found in the hollow part of most bones. Bone marrow aspiration is the removal of this tissue for examination. See also: Bone marrow biopsy Bone marrow culture How the Test is Performed     The health care provider will take the bone marrow from your pelvic or breast bone. (Occasionally, another bone is selected.) First, the area is cleaned with a germ-killing medicine, then numbing medicine (local anesthesia) is applied. Next, the health care provider inserts a special needle into the bone. The needle has a tube attached to it, which creates suction. A small sample of bone marrow fluid flows into the tube. The needle is removed. A laboratory specialist looks at the bone marrow fluid under a microscope. How to Prepare for the Test     No special preparation is necessary for this test. How the Test Will Feel     There will be a prick and a slight burning sensation with the local anesthetic. Pressure may be felt as the needle is inserted into the bone. There is a sharp sucking sensation as the marrow is aspirated, which lasts for only a few moments. Why the Test is Performed     This test is used to diagnose leukemia, infections, some types of anemia , and other blood disorders. It may help determine if cancers have spread. Normal Results     The marrow should contain blood-forming (hematopoietic) cells, fat cells, and connective tissues. What Abnormal Results Mean     Abnormal results may be due to: Acute lymphocytic leukemia Acute nonlymphocytic leukemia (AML) Anemia of B-12 deficiency Anemia of folate deficiency Chronic lymphocytic leukemia (CLL) Chronic myelogenous leukemia (CML) Idiopathic thrombocytopenic purpura (ITP) Lymphoma Macroglobulinemia of Waldenstrom Megaloblastic anemia Multiple myeloma Myelofibrosis Pernicious anemia Primary thrombocytopenia Risks     There may be some bleeding at the puncture site. More serious risks, such as serious bleeding or infection, are very rare. References     Hoffman R, Benz EJ, Shattil SS, et al. Hematology: Basic Principles and Practice . 4th ed. Orlando, Fl: Churchill Livingstone; 2005:2656-2657. Behrman RE. Nelson Textbook of Pediatrics. 17th ed. Philadelphia, Pa: WB Saunders; 2004; 1695-1697.
  • June 26, 2012 Schilling test: Alternative Names     Return to top Vitamin B12 absorption test Definition     Return to top The Schilling test is used to determine whether the body absorbs vitamin B12 normally. How the Test is Performed     Return to top You will get two doses of vitamin B12 (cobalamin). The first dose is radioactive and taken by mouth. The second dose is not radioactive and is given as a shot 2 - 6 hours later. The injection of vitamin B12 may sting. Your urine will then be collected over the next 24 hours to measure whether you are absorbing vitamin B12 normally. This test may be performed in four different stages to find the cause of low vitamin B12 levels. Stage I is as described above. If Stage I is abnormal, Stage II may be done 3 - 7 days later. In Stage II, you'll receive radioactive B12 along with intrinsic factor. Intrinsic factor is produced in the stomach and attaches (binds) to vitamin B12. Stage II of the test can tell whether low vitamin B12 levels are caused by problems in the stomach that prevent it from producing intrinsic factor. If a Stage II test is abnormal, a Stage III test is performed. In the Stage III test, the Stage II test is repeated after you have taken antibiotics for 2 weeks. This can tell whether abnormal bacterial growth has caused the low vitamin B12 levels. A Stage IV test determines whether low vitamin B12 levels are caused by problems with the pancreas. With this test, you will take pancreatic enzymes for three days, followed by a radioactive dose of vitamin B12. A 24-hour urine sample is needed. For adults: On day 1, urinate into the toilet after getting up in the morning. Collect all of your urine (in a special container) for the next 24 hours. On the morning of day 2, urinate into the container after getting up. The test is now complete. Cap the container. Keep it in the refrigerator or a cool place. Label the container with your name, the date, and the time you last urinated, and return it as instructed. For infants: Thoroughly wash the area around the area from which urine exits the body (urethra). Open a urine collection bag (a plastic bag with an adhesive paper on one end), and place it on your infant. For males, place the entire penis in the bag and attach the adhesive to the skin. For females, place the bag over the labia. Place a diaper over the infant (including the bag). Check the infant often and change the bag after the infant has urinated into it. For active infants, this procedure may take a couple of attempts -- lively infants can displace the bag, making it difficult to get the urine sample. Drain the urine into the container. Deliver the container to the laboratory or your health care provider as soon as possible after you've collected all of the urine. How to Prepare for the Test     Return to top Fast (you can drink water) for 8 hours before starting the test, then eat normally for the next 24 hours. The health care provider may ask you to stop taking drugs that can affect the test. You cannot have parenteral (intramuscular injection) B12 within 3 days before the exam. If the collection is being taken from an infant, you may need to use a couple of extra urine collection bags. How the Test Will Feel     Return to top The injection of vitamin B12 may sting. Why the Test is Performed     Return to top The Schilling test is performed to check vitamin B12 absorption. Intrinsic factor is produced in the stomach and is required for vitamin B12 absorption. If your body does not make intrinsic factor, you cannot absorb vitamin B12. The lack of intrinsic factor can lead to low levels of vitamin B12 because of pernicious anemia, partial removal of the stomach (gastrectomy), poor vitamin B12 absorption due to bowel disease, too much bacteria in the intestine, a lack of enough enzymes being produced by the pancreas, or certain medications. The Schilling test is most commonly used to evaluate patients for pernicious anemia. The test can be falsely positive. Most of the time this is due to poor urine collection. Other reasons include kidney disease or problems with the lining of the small intestine. Normal Results     Return to top Urinating 8 - 40% of the radioactive vitamin B12 within 24 hours is normal. What Abnormal Results Mean     Return to top Low vitamin B12 levels can cause pernicious anemia. This can occur if you have problems absorbing vitamin B12 or you don't eat enough foods that contain vitamin B12. Some other causes are removal of part of the stomach or the development of an antibody against intrinsic factor. If there is a problem with the stomach's ability to make intrinsic factor, Stage I of the test will be abnormal and Stage II will be normal. Both the Stage I and II Schilling tests will be abnormal in people who have problems absorbing vitamin B12 and intrinsic factor in the small intestine. Abnormal Stage I and II Schilling tests may indicate: Biliary disease Celiac disease (sprue) Hypothyroidism Liver disease Lower-than-normal amounts of vitamin B12 absorption may indicate: Biliary disease, resulting in poor absorption (malabsorption) of nutrients from the intestinal tract Intestinal malabsorption (for example, related to sprue or celiac disease) Liver disease (causing malabsorption) Pernicious anemia Additional conditions under which the test may be performed: Anemia of B12 deficiency Blind loop syndrome Megaloblastic anemia Risks     Return to top Local reaction to vitamin injection Nausea Feeling lightheaded
  • June 26, 2012 LDH: Alternative Names     Lactate dehydrogenase; Lactic acid dehydrogenase Definition     Return to top LDH is a blood test that measures the amount of lactate dehydrogenase (LDH). See also: LDH isoenzymes How the Test is Performed     Return to top The health care provider draws blood from a vein or from a heel, finger, toe, or earlobe. The laboratory quickly spins (centrifuges) the blood to separate the serum (liquid portion) from the cells. The LDH test is done on the serum. How to Prepare for the Test     Return to top Your health care provider may ask you to stop taking drugs that may affect the test. Drugs that can increase LDH measurements include anesthetics, aspirin, clofibrate, fluorides, mithramycin, narcotics, and procainamide. Why the Test is Performed     Return to top LDH is most often measured to check for tissue damage. The enzyme LDH is in many body tissues, especially the heart, liver, kidney, skeletal muscle, brain, blood cells, and lungs. LDH affects the chemical reaction for the conversion of pyruvate and lactate . Exercising muscles convert (and red blood cells metabolize ) glucose to lactate. Lactate is released into the blood and is later taken up by the liver. The liver converts lactate back to glucose and releases glucose into the blood. Resting muscles, red blood cells, and other tissues then take up this glucose. Normal Results     Return to top Normal values may vary slightly from one lab to another. A typical range is 105 - 333 IU/L (international units per liter). What Abnormal Results Mean     Return to top Higher-than-normal levels may indicate: Cerebrovascular accident (CVA, stroke) Heart attack Hemolytic anemia Low blood pressure Infectious mononucleosis Blood deficiency (intestinal ischemia) Liver disease (for example, hepatitis ) Muscle injury Muscular dystrophy New abnormal tissue formation (neoplastic) states Pancreatitis Tissue death (pulmonary infarction) If the LDH level is raised, your doctor may order an LDH isoenzymes measurement. Other conditions under which the test may be done: Anemia of vitamin B-12 deficiency Megaloblastic anemia Pernicious anemia References     Return to top Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB, McKenna WG. Clinical Oncology . 3rd ed. Philadelphia, Pa: Churchill Livingstone, 2004. Ferri FF. Ferri's Clinical Advisor 2007 . Philadelphia, Pa: Mosby, 2006.
  • June 26, 2012 Abstract There is evidence that vitamin A benefits hematopoiesis and iron metabolism. For this reason, we investigated the relationship of serum retinol concentration to hemoglobin concentration and biochemical indicators of iron status in Canadian aboriginal infants. No infant had biochemical evidence for vitamin A deficiency (serum retinol 0.35 mol/L) although 25.6% had anemia (hemoglobin 110 g/L) and 20.4% had iron deficiency (serum ferritin 10 g/L). Serum retinol concentration was significantly correlated with hemoglobin concentration (r 0.30, p 0.001, n 185) and serum iron concentration (r 0.44, p 0.001, n 166) but not with serum ferritin concentration. Anemic infants had lower serum retinol concentrations than those without anemia (1.09 0.33 mol/L vs. 1.36 0.36 mol/L, p 0.01). Infants given vitamin supplements that contained vitamin A, ascorbic acid, and vitamin D3 had a higher hemoglobin concentration than infants not given supplements (117 11 vs. 113 10 g/L, p 0.04) and a lower prevalence of anemia (21.9% vs. 37.0%, p 0.05). In conclusion, vitamin A was positively associated with hemoglobin concentration in this pediatric populatio
  • June 26, 2012 Abstract As part of a pig study to elucidate the interactions between low v itamin A status and helminth infections, surprisingly, we obser v ed higher haemoglobin le v els and packed cell v olumes in the pigs with low v itamin A status. A possible haemoconcentration effect, due to some disturbance in the regulation of the extracellular fluid v olume, could lead to underestimation of the pre v alence of anaemia in v itamin A deficient human populations. Therefore, this phenomenon needs to be further clarified in studies in v ol v ing determination of plasma v olumes. # 2002 Published by Elsevier Science B.V.
  • June 26, 2012 Vitamin E deficiency in the premature infant is associated with a hemolytic anemia. This anemia responds to tocopherol and the response is characterized by a rise in the hemoglobin and a fall in the reticulocyte count. Treatment of premature infants from birth wih supplemental vitamin E reduces the severity of anemia and prevents the marked reticulocytosis commonly observed in these infants of low birth weight. Vitamin E deficiency in the rat results in the appearance of a mild compensated hemolytic process. This is accompanied by thrombocytosis. The erythrocytes demonstrate increased pentose phosphate pathway activity. Tocopherol appears to be poorly absorbed from a low-fat diet in the premature infant. In infants with vitamin E deficiency there appears to be an increase in the creatine-to-creatinine ratio, lower serum proteins, and an increased excretion of methylmalonic acid in the absence of vitamin B 12 deficiency. The interrelationship of this potpourri of observations remains to be explained.
  • June 26, 2012 Abstract The high frequency of chromosomal breaks in Fanconi anemia (FA) lymphocytes has been related to the increased oxidative damage shown by these cells. The effect of 100 m M dl - a -tocopherol (Vitamin E) on the level of chromosomal damage in mitosis was studied in lymphocytes from five FA patients and from age matched controls, both under basal conditions and when G2 repair was prevented by 2.5mM caffeine (G2 unrepaired damage). In addition, the effect of this antioxidant on G2 duration and the efficiency of G2 repair was also evaluated in the sample. a -Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%). The efficiency of the G2 repair process (G2R rate) defined as the ratio between the percentage of chromatid breaks repaired in G2 and the duration of this cell cycle phase was lesser in FA cells (10.6) than in controls (22.6). AT treatment slightly increased this G2R rate, both in FA cells and controls. These results suggest that an increased oxidative damage and a lower G2 repair rate may be simultaneously involved in the high frequency of chromatid damage detected in FA cells. © 2001 Elsevier Science B.V. All rights reserved.
  • June 26, 2012 Effect of Vitamin E on î-AminolevulinicAcid Dehydratase Activity in Weanling Rabbits with Chronic Plumbism1 RICHARD S. BARTLETT, J. E. ROUSSEAU, JR., H. I. FRIER ANDR. C. HALL, JR. Department of Nutritional Sciences, University of Connecticut, Starrs, Connecticut 06268 ABSTRACT Lead inhibits several enzymes in the heme synthesis pathway with S-aminolevulinic acid dehydratase (ALAD) being the most sensitive. Vitamin E has a stimulatory effect on heme synthesis, apparently through its action on ALAD and on 5-aminolevulinic acid synthetase (ALAS). To study the possible effects of vitamin E in alleviating lead toxicity, rabbits were fed for 12 weeks a basal ration plus lead, either 25 mgAg body weight/day, or no lead, and one of four supplementary dl-a-tocopheryl acetate intakes equivalent to either 0, 1, 3, or 9 mg of itt-a-tocopherol/kg body weight/day. Equalized feeding of the basal ration was employed. Body weight was unaffected. In the lead-fed groups, plasma tocopherol was higher, whole blood lead concentration twice and liver lead four times greater. The concentration of liver lead increased 8 jug/100 g of fresh liver for each 10% increase in dietary tocopherol for those rabbits receiving vitamin E and lead. ALAD activity was depressed by lead; however, its activity was unaltered by vitamin E. Hematocrit levels were found to be lower in the plus-lead groups, incidence of basophilic stippling of erythrocytes greater, and urinary 5-aminolevulinic acid (UALA) and porphobilinogen greater. In rabbits fed lead, UALA concentration was increased by 1.02 mg ALA/100 ml for each 10% increase in vitamin E. Based upon these findings, it is suggested that vitamin E may have had an effect on the enzyme ALAS, rather than on ALAD. J. Nutr. 104: 1637-1645, 1974. INDEXING KEYWORDS vitamin E •plumbism •rabbits Plumbism has been found to produce Of these enzymes, ALAD was observed to several disorders (1). These included be the most sensitive to lead (1, 3). This peripheral neuropathy, encephalopathy, enzyme is essential not only for hemoacid- fast intranuclear inclusion bodies in globin formation but also for the synthesis the kidney, specific abnormalities in the of heme-containing respiratory enzymes, heme synthesis pathway, alterations in red among them catalase, the cytochromes blood cell morphology, and anemia. P-450, b, c, and c\\ as well as the cyto- Several enzymes in the heme synthesis chromes a and c3, which contain modified pathway have been reported to be inhib- heme as a prosthetic group (3). ited by lead: S-aminolevulinic acid syn- Dinning and Day (4) observed a nutrithetase (ALAS) (EC 2.3.1.37) (2), which tional anemia in primates fed a vitamin catalyzes the reaction of glycine and sue- E-deficient diet and thus indicated a cinyl CoA to 8-aminolevnlinic acid (ALA), possible involvement of the vitamin in the 8-aminolevulinic acid dehydratase (ALAD) synthesis of heme or heme proteins. Sub- (EC 4.2.1.24) (1, 3), which catalyzes the sequently, Nair et al. (5, 6) conducted reaction of 2-ALA to porphobilinogen (PEG), and heme synthetase, which is Receivedfor publicationMayso, 1974. i-ortniiwl fnr f-tif»inr-nrnnraHnn nf trip fpr- l Scientific Contribution no. 886, Storrs Agricul- required tor trie incorporation or tne rer tural Experlment statloni university of Connecticut, rous ion into the porphyrm ring structure, storrs, conn. 1637 at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from 1638 BARTLETT, ROUSSEAU, FRIER AND HALL studies to determine the relationship of vitamin E to heme synthesis. One conclu sion of these studies was that a-tocopherol has a slight stimulatory effect on the activ ity of ALAS and ALAD. deRosa (7) added vitamin E to the diets of lead-intoxicated rabbits and found a diminution in the anemia and coproporphyrinuria, which had resulted from plumbism. To study the possible effects of tocopherol on the inhibition of ALAD activity by lead, rabbits were fed either a ration free of added lead, or the same ration plus an intake of lead sufficient to produce mild toxicity. In addition, rabbits received graded intakes of vitamin E ranging from deficient to nine times their estimated re quirement. METHODS Animals. Thirty-two 31-day-old male New Zealand rabbits, in four sets of eight were obtained over a 4-week period, num bered at random, and placed in individual stainless-steel cages, 51 cm wide X 56 cm long X 38 cm high, with a 1.25-cm mesh no. 12 wire floor. The feeding regimen con sisted of a 1-week transition period in which the rabbits were fed 100 g of a basal ration plus decreasing amounts of hay, a 2-week standardization period in which the rabbits were fed basal ration only, and a 12-week comparison period in which the rabbits were fed basal ration plus supple ments. The supplements provided either 0 or 25 mg Pb/kg body weight/day and one of four, 0, 1, 3, or 9 mg cß-a-tocopherol/kg bodv weight/day. The basal ration consisted of, in kg/100 kg: 20.590,462 ground barley, 22.5 ground oats, 6.75 solvent extracted linseed oil meal, 6.75 solvent process soybean oil meal 50%, 10 ground dried beet pulp, 22.5 ground wheat bran, 9 cane molasses, 0.75 calcium carbonate, 0.25 steamed bone meal, 0.9 iodized sodium chloride, 0.001,538 vitamin A supplement,2 and 0.008 irradiated yeast3 and was pelleted with pellets being 0.5 cm in diameter. The amount of basal ration offered was identical to that previously de scribed (8). The added dietary lead was fed as lead acetate, Pb(Ac)2-3H2O,* in basal ration pellets at a concentration of 10 mg Pb/g. The added dietary vitamin E was fed as (ii-a-tocopheryl acetates in basal ration pellets containing the equiv alent of 0.5 mg dZ-a-tocopherol/g. The level of lead was chosen to produce a mild toxicity in rabbits and was based on a previous study6 in which urinary ALA con centration was elevated. The levels of a-tocopherol equivalent were chosen to produce a deficiency, 0-intake group, to meet the rabbit's requirement (9), 1 mg, and to provide levels, 3 and 9 mg, in excess of the established requirement. Based on a calculated content of 11 mg tocopherol/kg of basal ration (10, 11) and on feed con sumption data (8), rabbits in the 0-intake group were expected to have an intake of tocopherol equal to 0.5 mg/kg body weight/day. Both supplementary lead and supplementary vitamin E were fed at 11 AM. After each rabbit had completely consumed its respective supplements, the daily allowance of the basal ration (minus the weight of the supplements) was fed. Observations and analyses. All feed of fered and refused daily was weighed to the nearest gram. A sample of the basal ration was analyzed for lead (12) and found to contain 0.002 mg Pb/g. Rabbits were weighed upon arrival and on the morning of the last day of each experi mental week, as well as on the morning of killing. The mean ± standard error for the minimum and maximum ambient tempera tures during the comparison period were 19.9 ±0.2 and 24.1 ±0.1°,respectively. Light intensity, limited to incandescent sources within the animal room and mea sured every 4 weeks, averaged 161 ±9 lux. At 8 AMon day 1 of weeks 1, 7, and 13 of the comparison period, heparinized blood samples were obtained by ear punc ture (13). Whole blood was analyzed for hematocrit, ALAD activity (14), and lead concentration (12), but the latter determi nation was omitted in the case of the *Hoffmann-La Roche type 325-40 vitamin A acetate boadlets, 325.000 U.S.P. unlts/g, contributes 1,500 UK of retlnol equivalent/kg of ration. »Standard Brands type 36-F, 16.000.000 IU vitamin D per pound, contributes 2.880 IÃoevitamin D/kg of ration. ' Lead acetate, Merck, reagent crystals, item no. 7408. and lot no. 72700. 5Hoffmann-La Roche, dry vitamin B acetate 50%. containing not less than 500 IÃoeof di-«-tocophervl acetate. N.F. per gram, lot no. 025081. «Roscoe.D. B. (1973) The clinical pathology of rabbits suffering from Induced chronic pliimblsm and a comparison of three diagnostic tests. M.S. thesis. University of Connecticut, Storrs, Conn. at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from VITAMIN E AND PLUMBISM IN RABBITS 1639 TABLE 1 Feed consumption anil growth (tola for rabbits receiving no lead or lead and graded doses of vitamin K Added a-tocopherol equivalent1 Criterion Pb» SD per rabbit mg/kg body wt/dayRabbits (no.)Feed consumed (kg) —Body wt (kg) InitialTerminal449.8(9.1)3 9.1(9.7)1.5 1.23.8(3.5)3 3.6(3.8)4 39.1(9.0) 8.4(9.0)1.31.23.4(3.4) 3.2(3.4)3410.3(9.4) 9.1(9.5)1.5 1.23.9(3.6) 3.5(3.6)3410.4(9.5) 8.0(9.0)1.51.13.8(3.5) 3.1(3.4)—1.4(0.9)0.20.5(0.3) 1As d¡-a-tocopherylacétate. initial body weight. 2 —= no added lead; + = 25 mg Pb/kg body weight/day. 'Adjusted by covariance for week-1 sample. Plasma obtained by centrifugation was analyzed for tocopherol content (15). At weeks 7 and 13, blood smears were made and stained with Wright's stain to be examined for basophilic stippling (16). On the last day of weeks 6 and 12 of the comparison period, rabbits were placed in individual metabolism cages at 7:45 AM. Urine was collected in jars containing 5 ml of tartaric acid preservative ( 17). The first 24-hour sample was discarded. The second 24-hour sample was measured for volume. From this sample, a 10-ml subsample was obtained, brought to a pH of 5-7 with sodium acetate, and analyzed for ALA and PEG (18). At 8 AMon day 1 of week 13 of the com parison period, the rabbits were weighed, anesthetized with sodium pentobarbital, and decapitated. Samples were taken from the biceps femoris, psoas major, subscapularis, and larynx and placed in 10% for malin. Histological sections from these muscles were stained with hematoxylin and eosin and examined for the presence or absence of muscular dystrophy. The liver was also removed, weighed, homog enized in a Waring blender, refrigerated, and subsequently analyzed for dry matter, ash, and lead (12). The data were subjected to analysis of variance and covariance (19), the latter where applicable, so as to isolate the vari ation due to dietary treatments and to rabbits within treatments. In addition, the variation due to treatments was further partitioned into contrasts between no lead versus added lead and among added di etary tocopherol intakes, within either minus lead or plus lead. The variation for among dietary tocopherol intakes within either minus or plus lead was divided into 0 versus 1, 3, or 9 supplementary tocoph erol intakes and among added tocopherol. The among added tocopherol was further divided into linear and residual compo nents (linear regression of response on logjo a-tocopherol equivalent intake). The standard deviation (so) per rabbit, pre sented in the tables, was from the analysis of variance and equaled the square root of the mean square for rabbits within treat ments. In the case of liver lead concentra tion, ALAD activities and urinary ALA and PEG concentrations and daily outputs, the variances for those groups receiving supplemental lead were considerably greater than for those receiving no lead. Therefore, SD'S are given for groups fed either minus or plus lead, and Cochran's test of significance (19) for two samples with unequal variance was used. Three rabbits died during the experimental period, two of unknown causes, one of these prior to beginning the comparison period and the other in week 4 of the com parison period. The third died while being bled at the onset of the comparison period. Data obtained from these rabbits were not included in the computation of results. at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from 1640 BARTLETT, ROUSSEAU, FRIER AND HALL TABLE 2 Plasma tocopherol and whole blood lead values for rabbits receiving no lead or lead and graded doses of vitamin E Added a-tocopherol equivalent1CriterionPlasma tocopherol (/ig/100 ml)Actual valuesInitialWeek?TerminalMean (weeks 7 and terminal)InitialWeek?TerminalMean (weeks 7 and terminal)Whole blood Pb Gig/100 ml)Week?TerminalMeanPb« 0l398D per rabbitmg/kg body wt/day346+ 387- 205 +367- 164 + 394184 +3802.51+ 2.572.30(2.28)' +2.58(2.52)- 2.16(2.14) + 2.59(2.52)2.24(2.22) +2.58(2.53)- 29+ 9045 + 9837 + 94220 369654 902489 875571sss2.34 2.562.80(2.87) 2.95(2.92)2.67(2.79) 2.93(2.87)2.74(2.84) 2.95(2.90)4213744 9643 117378 320883 1,216978 1,160930 1,1882.55 2.482.94(2.91) 3.05(3.06)2.96(2.90) 3.05(3.06)2.96(2.92) 3.06(3.06)36945210644 100294 2941,660 1,7881,732 1,7401,690 1,7(>42.45 2.463.21 (3.23) 3.24(3.26)3.20(3.23) 3.23(3.25)3.21(3.23) 3.24(3.25)3810845 11541 1110.140.11(0.10)0.17(0.13)0.12(0.09)241727 1As di-a-tocopheryl acetate. * ——no added lead; + »25 mg Pb/kg body weight/day. «so of means of actual values increased with magnitude of means, therefore, individual values transformed to their respective logarithms to the base 10. *Ad justed by covariance for initial tocopherol levels. RESULTS Feed consumption and body weight were not significantly different among treat ments (table 1). During the comparison period, these rabbits were offered an aver age of 9.5 kg of ration with a so = 1.4 and gained in body weight 2.2 kg with the so = 0.4. Adjusted plasma tocopherol concentra tion (table 2), when expressed on a Iog10 basis of /¿g/100ml, YI, was found to in crease linearly with increasing added levels, logic basis of 1, 3, and 9 mg/kg body weight/day, of dietary a-tocopherol equiv alent, X. The regressions for the minusand plus-lead groups were as follows: week 7: -, FI = 2.83 + 0.37 X ±0.10 + , Ft = 2.91 +0.35X ±0.10 terminal: -, FI = 2.76 + 0.46X ±0.13 + , Fi = 2.87 + 0.40X ±0.13 mean: -, F! = 2.80 + 0.41 X ±0.09 +, F, = 2.90 + 0.37 X ±0.09 The concentrations of tocopherol in the plasma of those rabbits receiving supple mentary lead were higher than the concen trations in the plasma of rabbits receiving no lead (P < 0.001); however, the linear responses were found not to be signifi cantly different. at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from VITAMIN E AND PLUMBISM IN RABBITS 1641 TABLE 3 Liver weight and lead concentration for rabbits receiving no lead or lead and graded doses of vitamin E Added a-tocopherol equivalent1Criterion Pb!Liver wtTotal (g)Per unit body wt (g/kg)Dry matter (g/100 g fresh) -Ash (g/100 g fresh)0i39so per rabbitmy/ku body wt/day104 11628 3327.4 26.91.14 1.1377 9223 3027.827.51.18 1.2395 10025 3027.8 27.81.14 1.1697 10028 3427.8 27.11.09 1.131860.90.04 Liver Pb Fresh (pg Pb/100 g) Dry matter (mg Pb/100 g) Ash (mg Pb/100 g) 52 386 0.19 1.44 4 :¡4 82 394 0.30 1.43 7 32 61 426 0.22 1.54 5 36 56 568 0.20 2.12 5 50 24 84 0.09 0.33 27 i As Ãà ¼-0-tocopheryl acetate. ! —= no added lead ; + = 25 mg Pb/kg body weight/day. Blood and liver lead concentrations, tables 2 and 3, were, as expected, greater in the plus-lead groups, P < 0.001. Liver weight per unit body weight was greater in the plus-lead rabbits, P < 0.001. Al though vitamin E had no significant effect on blood lead concentration, it did affect the lead concentration in the livers of rab bits receiving added lead. Regressions of liver lead concentration in mg/g, Ya, on the logio of supplementary a-tocopherol equiv alent, X, for those rabbits receiving plus lead were : fresh: F2 = 0.37 + 0.190 X ±0.08 dry matter : F2 = 1.33 + 0.749 X ±0.33 ash: F2 = 30 + 19.47 X ±7 A 10% increase in added dietary vitamin E resulted in an increase of 0.008 mg Pb/100 g fresh liver (P < 0.001), 0.031 mg Pb/100 g dry matter (P<0.05), and 0.80 mg Pb/100 g ash (P < 0.01). Hematological findings, indicative of lead toxicity, were lower hematocrit and ALAD activity and greater basophilic stip pling (table 4). There was a significantly lower hematocrit in the plus-lead groups seen at the 7th (P<0.05) and terminal weeks (P < 0.01 ) and for the average of these two (P<0.01). ALAD activity was similarly less due to added lead with P < 0.001 for week 7, terminal, and mean values. There was a significantly larger number of stippled red cells (P<0.10) in the rabbits fed supplemental lead than in those fed no supplemental lead. None of these criteria showed any consistent posi tive or negative trend with increasing vitamin E intake. Urinary volume, ALA concentration and total output, and PEG concentration and total output are presented in table 5. There were no significant effects of treat ments on urine volume. Rabbits receiving lead had significantly greater urinary con centrations (P< 0.001) and total daily outputs (P< 0.001) of both ALA and PEG than did rabbits receiving no lead. In rabbits receiving lead, added dietary vitamin E resulted in linear increases in urinary ALA concentrations expressed as mg/100 ml, Y3, and daily output expressed as mg/day, Y4. Linear regressions of these responses, Y3 and Y4, on the logio of added dietary a-tocopherol equivalent, X, were at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from ir>42 BARTLETT, ROUSSEAU, FRIER AND HALL for urinary ALA concentration : weck 6: Y3 = 7.40 + 8.26 X ±4.2 week 12 : Y3 = 4.04 + 10.50 X ±4.4 mean: Y3 = 5.63 + 9.55X ±5.8 and for total daily urinary output of ALA: week 6: F4 = 10.71 + 11.52 X ±7.9 week 12: F4 = 7.46 + 10.71 X ±2.9 mean: F4 = 9.09 + 11.13X ±4.7 A 10% increase in added dietary vitamin E brought about an increase in urinary ALA concentration of 0.80 mg/100 ml urine for week 6 (P<0.05), 1.02 mg/100 ml urine for week 12 (P <0.05), and 0.93 mg/100 ml urine for the mean (P < 0.10). Similarly a 10% increase in added dietary tocopherol brought about an increase in the total daily output of ALA of 1.12 mg/ day for week 6 (P<0.10), 1.04 mg/day for week 12 (P<0.01), and 1.08 mg/day for the mean of these (P <0.05). Muscular dystrophy was seen in one rabbit that received 25 mg Pb and 9 mg tocopherol/kg body weight/day and that exhibited edema and moderate fiber de generation of laryngeal muscles. DISCUSSION In the present study, vitamin E did not prevent a decrease of ALAD activity in lead-fed rabbits. Furthermore, vitamin E had no stimulatory effect on ALAD in rabbits fed no lead. Our discrepancy with Nair et al. (5), who reported that tocoph erol stimulated the enzyme, may be due to the mode of tocopherol administration. Nair et al. fed a single oral dose of 100 mg vitamin E per rat. Our rabbits were fed much smaller doses over a longer period of TABLE 4 Hemalocril, erythrocyle &-aminolevidinic acid dehydratase (ALAD) activity, and basophilic stippling for rabbits receiving no lead or lead and graded doses of vitamin E Added a-tocopherol equivalent1CriterionHematocrit (%)InitialWeek 7TerminalMean (weeks 7 and terminal)Erythrocyte ALAD activity, unit«4Week 7TerminalMeanBasophilic stippling ofWeek 7TerminalMeanPb» 0l398 Dper rabbitmy/kg body wt/day- 44.9 + 41.741.2(40.4)" + 36.7(37.5)44.0(43.2) + 37.7(38.3)42.6(41.8) + 37.2(37.8)14.5 + 0.921.7 + 1.118.1 + 1.0red blood cells0.3(1.1)' + 0.3(1.1)0.0(1.0) + 3.8(1.8)0.1(1.1) + 2.3(1.6)41.3 43.442.6(43.5) 41.7(41.6)42.2(43.0) 43.1(43.0)42.4(43.2) 42.4(42.2)20.1 1.118.0 0.419.0 0.80.0(1.0) 0.5(1.2)0.0(1.0) 0.0(1.0)0.0(1.0) 0.2(1.1)43.6 42.944.4(44.2) 39.2(39.4)45.7(45.5) 41.0(41.2)45.1(44.9) 40.2(40.3)20.0 0.825.7 0.822.9 0.80.0(1.0) 1.3(1.5)0.0(1.0) 0.2(1.1)0.0(1.0) 0.7(1.3)42.6 45.039.9(40.2) 38.4(37.4)44.8(45.1) 41.6(40.8)42.4(42.7) 40.0(39.2)17.7 0.715.11.715.9 1.20.0(1.0) 0.0(1.0)0.0(1.0) 0.7(1.2)0.0(1.0) 0.3(1.1)2.93.2(3.0)2.5(2.3)2.7(2.4)6.5 0.88.00.57.0 0.4— (0.2)-(0.5)-(0.3) 1As dZ-a-tocopheryl acetate. ! —= no added lead ; + = 25 mg Pb/kg body weight/day. ' Adjusted by covariance for initial hematocrit. ' One unit ALAD activity = amount of ALAD necessary to convert 1 mole/ml/min ALA to PBG/ml RBC. • Vinumber stippled cells/100 RBC) + 1. at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from VITAMIN E AND PLUMBISM IN RABBITS 1Ãoe43 TABLE 5 Urinary volume, &~aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations and daily outputs for rabbits receiving no lead or lead and graded doses of vitamin E Added a-tocopherol equivalent1Criterion Pb'Urine volume (ml)Week 6Week 12MeanUrinary ALA concentration (mg/100 ml)Week 6Week 12MeanTotal output (mg/day)Week 6Week 12MeanUrinary PBG concentration (iig/100 ml)Week 6Week 12MeanTotal output (/ig/day)Week 6Week 12Mean0l39so per rabbitmg/ky body wt/day290 200200 210240 2000.12 1.260.20 0.920.16 1.090.32 1.770.40 1.560.36 1.66668376947867657160 1,346134 914148 1,130160 180130 220150 2000.15 0.750.14 0.280.14 0.520.21 1.240.18 0.670.19 0.9694 77683 26688 5211381,228126 577132 903190 130130 130160 1300.14 1.120.18 1.080.16 1.100.27 1.360.18 1.370.25 1.3755 965106 60081782106 1,163143742124 953200 180180 170190 1700.15 1.540.18 1.320.16 1.440.31 2.300.31 1.710.31 2.0155862107 64781 7541131,416182872147 1,1449080700.07 0.420.07 0.440.05 0.580.12 0.790.20 0.350.10 0.4730 41743 3133132052 59480 39463 365 ' As di-a-tocopheryl acetate. * —= no added lead ; + = 25 mg Pb/kg body weight/day. time. It is possible that, although we were feeding excessive tocopherol, it was not of sufficient magnitude to reproduce the find ings of Nair et al. Similarly deRosa (7 ) fed relatively high doses of vitamin E, 100 mg, to lead-intoxicated rabbits. The fact that ALAD, the enzyme re sponsible for converting ALA to PBG, is inhibited in lead poisoning has been sug gested as a possible causative factor for the increase of urinary ALA concentration. As expected, lead-fed rabbits excreted greater amounts of ALA in the urine. How ever, we also found that urinary ALA in creased with increasing amounts of supple mental vitamin E. The most logical ex planation for this would be that vitamin E may have a stimulatory effect on the en at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from 10-4-1 BARTLETT, ROUSSEAU, FRIER AND HALL zyme ALAS, which catalyzes the conden sation reaction of glycine plus succinyl CoA to ALA. This would bring about a graded increase in ALA production, but with ALAD being inhibited by lead, the ALA would be produced in excess and excreted in the urine. Nair et al. (5) found that vitamin E induced a significant rise in the activity of ALAS in control rats. How ever, because we did not measure ALAS activity in either group, we can only sup port this finding by inference. Although ALAD was inhibited in the plus-lead groups, a significant increase in urinary PEG was still noted, which is in agreement with the findings of others ( 16, 20). An explanation for such an effect is not clearly evident, because with low levels of ALAD activity, it would seem likely that only small quantities of PEG would be formed. A possibility is that an enzyme or some combination of enzymes between PEG and heme is inhibited by lead causing a buildup of heme precursors. The enzyme, heme synthetase, has been shown to be in hibited by lead (20), and there is sug gestive evidence that the enzymes uroporphyrinogen decarboxylase and coproporphyrinogen decarboxylase are also inhibited by lead (21). It has also been suggested that the increase of heme syn thesis reactants in the urine may be caused by hemoglobin degradation due to lead poisoning. However, Grinstein et al. (22), using 16N-labeled glycine, have shown that this does not seem to be the case in leadintoxicated rabbits. The decrease in hematocrit and in creased incidence of stippling of erythrocytes were expected. Exposure to lead has been shown to increase the mechanical fragility of erythrocytes (23) and shorten survival times of red cells (24). Stippling of erythrocytes is commonly found in lead poisoning, although it alone is not con sidered a good indicator of lead toxicity because of its lack of specificity (23) and its poor correlation with other criteria in dicative of plumbism (16, 25 ). Possible explanations as to why plasma tocopherol was greater in the lead-fed rab bits would involve increased absorption and/or the release of tocopherol into the circulatory system. Bile and pancreatic juices have an obligatory role in the ab sorption of a-tocopheryl acetate (26), and it has been proposed that pancreatic juice may contain an o-tocopherol ester hydrolase enzyme, which would catalyze the hydrolysis of a-tocopheryl acetate, a re action believed necessary for optimal ab sorption. Injected doses of lead have been shown to increase biliary flow (27). There fore, increased biliary flow due to lead might increase absorption of a-tocopheryl acetate. Another way in which lead could have increased absorption of a-tocopheryl acetate would be through stimulation of the a-tocopherol ester hydrolase by lead. It is also possible that because approxi mately one-third of the circulatory a-to copherol is bound to the erythrocytes (28), and excessive intakes of lead result in greater mechanical fragility of erythro cytes (23) and alteration of membrane permeability (29), bound vitamin E was freed to the plasma in the lead-intoxicated rabbits. Our negative finding concerning mus cular dystrophy was probably due to sev eral factors. First, the mean plasma tocoph erol levels at the onset of the comparison period were quite high, 324 /¿g/100ml, suggesting high initial vitamin E stores in the rabbits. Second, the basal ration was calculated to supply each rabbit with 0.5 mg/kg body weight/day, one-half of their minimum daily requirement (9). There fore, with high initial stores of vitamin E and with a small amount of this vitamin available in the basal ration, rabbits fed no added vitamin E were not sufficiently deficient to develop muscular dystrophy. Because red blood cell hemolysis in sev eral species, rat (28), dog (30), man (31), and rabbit (32), occurs at plasma tocoph erol levels of 300-500 /ng/100 ml, the rab bits fed no supplementary tocopherol could be considered deficient to borderline with respect to vitamin E deficiency. There was no apparent explanation for tocopherol's effect of increasing the liver concentration of lead. Information neces sary to understand this would include to copherol's relationship to the absorption, deposition, and excretion of lead. ACKNOWLEDGMENTS The authors wish to thank Drs. H. D. Eaton and S. W. Nielsen for their aid in at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from VITAMIN E AND PLUMBISM IN RABBITS 1645 preparation of the manuscript, B. A. Donohue and T. Watts, Sr., for care of the animals, Mrs. Frances Nichols for able technical assistance, and Marjorie Wood ward for aid in the absorption spectrophotometric determination of lead. LITERATURE CITED 1. Goyer R. A. & Rhyne, B. C. (1973) Patho logical effects of lead. Int. Rev. Exp. Pathol. 12, 1-77. 2. Kao, R. L. C. & Forbes, R. M. (1973) Ef fects of lead on heme-synthesizing enzymes and urinary delta-aminolevulinic acid in the rat. Proc. Soc. Exp. Biol. Med. 143, 234-237. 3. Hernberg, S. & Nikkanen, J. (1972) Effect of lead on delta-aminolevulinic acid dehydratase— a selective review. Prac. Lek. 24, 77-83. 4. Dinning, J. S. & Day, P. L. (1957) Vitamin E deficiency in the monkey. J. Exp. Med. 105, 395-aOl. 5. Nair, P. P., Murty, H. S. & Grossman, N. R. (1970) The in vivo effect of vitamin E in experimental porphyria. Biochim. Biophys. Acta 215, 112-118. 6. Nair, P. P., Murty, H. S., Caasi, P. I., Brooks, S. K. & Quartner, J. (1972) Vitamin E. Regulation of the biosynthesis of porphyrins and heme. J. Agr. Food Chem. 20, 476-480. 7. deRosa, R. (1954) L'azione dell'alfa-tocof erolo nella intossicazione sperimentale da piombo. Comportamento della coproporfirnuria e della crasi ematica. Acta Vitaminol. 8, 167- 172. 8. Hall, R. C., Jr., Frier, H. L, Bartlett, R. S. & Rousseau, J. E., Jr. (1974) Cerebrospinal fluid pressure in weanling rabbits with chronic plumbism. University of Connecticut, Storrs Agricultural Experiment Station Research Re port 43. 9. National Academy of Sciences-National Re search Council (1966) Nutrient Require ments of Rabbits. NAS-NRC publication 1194, Washington, D. C. 10. National Academy of Sciences-National Re search Council (1971) Adas of Nutritional Data on United States and Canadian Feeds. NAS-NRC, Washington, D. C. 11. National Academy of Sciences-National Re search Council (1969) United States- Canadian Tables of Feed Composition. NASNRC publication 1684, Washington, D. C. 12. Dalton, E. F. & Malonski, A. J. (1969) Atomic absorption analysis of copper and lead in meat and meat products. J. Ass. Offic. Anal. Chem. 52, 1035-1038. 13. Hoppe, P. C., Laird, C. W. & Fox R. R. (1969) A simple technique for bleeding the rabbit ear vein. Lab. Anim. Care 19, 524— 525. 14. Weissberg, J. B., Lipschutz, F. & Oski, F. A. (1971) delta-Aminolevulinic acid dehydratase activity in circulating blood cells. New Engl. J. Med. 284, 565-569. 15. Ouaife, M. L. & Harris, P. L. (1944) The chemical estimation of tocopherols in blood plasma. J. Biol. Chem. 156, 499-505. 16. Haeger-Aronsen, B. (1960) Studies on urinary excretion of delta-aminolevulinic acid and other haem precursors in lead workers and lead-intoxicated rabbits. Scand. J. Clin. Lab. Invest. 12 (suppl. 47), 1-128. 17. Vincent, W. F. & Ullman, W. W. (1970) The preservation of urine specimens for delta-aminolevulinic acid determination. Clin. Chem. 16, 612-613. 18. Mauzerall, D. & Granick, S. (1956) The occurrence and determination of delta-amino levulinic acid and porphobilinogen in urine. J. Biol. Chem. 219, 435-446. 19. Snedecor, G. W. & Cochran, W. G. (1967) Statistical Methods, 6th ed. Chapters 4, 10, 11, 12, and 14. Iowa State Univ. Press, Ames. 20. Gibson, S. L. M. & Goldberg, A. (197Ç) Defects in haem synthesis in mammalian tis sues in experimental lead poisoning and ex perimental porphyria. Clin. Sci. 38, 63-72. 21. Kreimer-Birnbaum, M. & Grinstein, M. ( 1965 ) Porphyrin biosynthesis. III. Porphyrin metabolism in experimental lead poisoning. Biochim. Biophys. Acta 111, 110-123. 22. Grinstein, M., Wikoff, H. H., de Mello, R. P. & Watson, C. T. (1950) Isotopie studies of porphyrin and hemoglobin metabolism. II. The biosynthesis of coproporphyrin III in experi mental lead poisoning. J. Biol. Chem. 182, 723-726. 23. Hass, G. M., Brown, D. V. L., Eisenstein, R. and Hemmens, A. (1964) Relationships between lead poisoning in rabbit and man. Amer. J. Pathol. 45, 691-728. 24. Hernberg, S., Nurminen, M. and Hasan, J. (1967) Nonrandom shortening of red cell survival times in men exposed to lead. En viron. Res. 1, 247-261. 25. de Bruin, A. & Hoolboom, H. (1967) Early signs of lead-exposure—a comparative study of laboratory tests. Brit. J. Ind. Med. 24, 203- 212 26. Galïo-Torres, H. E. (1970) Obligatory role of bile for the intestinal absorption of vitamin E. Lipids 5, 379-384. 27. Blaxter, K. L. (1950) Lead as a nutritional hazard to farm livestock. II. The absorption and excretion of lead by sheep and rabbits. J. Comp. Pathol. 60, 140-159. 28. Bieri, J. G. & Poukka, R. K. H. (1970) In vitro hemolysis as related to rat erythrocyte content of a-tocopherol and polyunsaturated fatty acids. J. Nutr. 100, 557-564. 29. Barltrop, D. & Smith, A. (1971) Inter action of lead with erythrocytes. Experientia 27 92—93 30. Hayes, K.' C. & Rousseau, J. E., Jr. (1970) Dialuric acid hemolysis as an index of plasma tocopherol concentrations in the dog. Lab. Anim. Care 20, 48-51. 31. Leonard, P. J. & Losowsky, M. S. (1967) Relationship between plasma vitamin E level and peroxide hemolysis test in human subjects. Amer. J. Clin. Nutr. 20, 795-798. 32. Horn, L. R., Barker, M. O., Reed, G. & Brin, M. (1974) Studies on peroxidative hemoly sis and erythrocyte fatty acids in the rabbit: effect of dietary PUFA and vitamin E. J. Nutr. 104, 192-201. at Isfahan University of Medical Sciences on January 5, 2008 jn.nutrition.org Downloaded from
  • June 26, 2012 . Figure 1 illustrates some of the basic features of iron metabolism and erythropoiesis, emphasizing points in the process at which certain vitamins may influence iron deficiency and anaemia. Vitamins such as vitamin A, folic acid, vitamin B 12 , riboflavin and vitamin B 6 , are necessary for the normal production of red blood cells, while others such as vitamins C and E protect mature red blood cells from premature destruction by free radical oxidation ( Table 2 ). Riboflavin, vitamin A and vitamin C may also prevent anaemia by improving intestinal absorption of iron, or by facilitating its mobilization from body stores. This paper explores the effects of these vitamins in the treatment and prevention of anaemia in human populations and identifies areas for future research.
  • June 26, 2012 QUICK SEARCH:   [advanced] Author: Keyword(s): Year: Vol: Page:  Institution: Isfahan University of Medical Sciences | Sign In via User Name/Password This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Atamna, H. Articles by Ames, B. N. Search for Related Content PubMed PubMed Citation Articles by Atamna, H. Articles by Ames, B. N. © 2007 The American Society for Nutrition J. Nutr. 137:25-30, January 2007 Biochemical, Molecular, and Genetic Mechanisms Biotin Deficiency Inhibits Heme Synthesis and Impairs Mitochondria in Human Lung Fibroblasts 1 Hani Atamna*, Justin Newberry, Ronit Erlitzki, Carla S. Schultz and Bruce N. Ames* Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute (CHORI), Oakland, CA 94609 * To whom correspondence should be addressed. E-mail: [email_address] or [email_address] .    ABSTRACT TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED   Four of the 5 biotin-dependent carboxylases (BDC) are in the mitochondria. BDC replace intermediates in the Krebs [tricarboxylic acid (TCA)] cycle that are regularly removed for the synthesis of key metabolites such as heme or amino acids. Heme, unlike amino acids, is not recycled to regenerate these intermediates, is not utilized from the diet, and must be synthesized in situ. We studied whether biotin deficiency (BD) lowers heme synthesis and whether mitochondria would be disrupted. Biotin-deficient medium was prepared by using bovine serum stripped of biotin with charcoal/dextran or avidin. Biotin-deficient primary human lung fibroblasts (IMR90) lost their BDC and senesced before biotin-sufficient cells. BD caused heme deficiency; there was a decrease in heme content and heme synthesis, and biotin-deficient cells selectively lost mitochondrial complex IV, which contains heme-a. Loss of complex IV, which is part of the electron transport chain, triggered oxidant release and oxidative damage, hallmarks of heme deficiency. Restoring biotin to the biotin-deficient medium prevented the above changes. Old cells were more susceptible to biotin shortage than young cells. These findings highlight the biochemical connection among biotin, heme, and iron metabolism, and the mitochondria, due to the role of biotin in maintaining the biochemical integrity of the TCA cycle. The findings are discussed in relation to aging and birth defects in humans.    Introduction TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED   Biotin is one of the least-studied vitamins, particularly in relation to mitochondrial function and the extent of its nutritional deficiency in humans ( 1 ). Biotin deficiency (BD) 2 in humans appears more common than previously thought ( 2 ), as is marginal deficiency during human pregnancy ( 2 ). Marginal BD in rodents is teratogenic even though the dams appear normal ( 2 , 3 ). The high biotin requirement of the developing human fetus may explain the susceptibility of embryonic development to BD ( 4 ). Although the role of biotin in mitochondrial biochemistry is well established ( 5 ), few studies of BD have focused on how such a deficiency affects mitochondria ( 6 ), oxidative stress, and aging ( 7 ). Biotin is a coenzyme in 5 different biotin-dependent carboxylases (BDC), which catalyze carboxylation reactions ( 5 ): pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), acetyl-CoA carboxylase (ACC)-2, and ACC-1. The first 4 are located in the mitochondria. PC, PCC, and MCC catalyze anaplerotic reactions and replenish tricarboxylic acid (TCA) cycle intermediates ( 8 ). The fifth BDC, ACC-1, is located in the cytosol and is important for fatty acid metabolism, as is ACC-2. The carboxyl group of carboxy-biotin is transferred by PC to pyruvate to form oxalacetate; by PCC to propionyl-CoA to form succinyl-CoA; and by MCC to 3-methylcrotonyl-CoA to form 3-methyglutaconyl-CoA, which is metabolized to acetyl-CoA ( 5 ). All feed directly into the TCA cycle ( 5 ). This study examines the effects of BD on mitochondria and cellular senescence resulting from modulations of anaplerotic carboxylation reactions. BD has a detrimental effect on the level of TCA cycle intermediates. A deficiency in PC directly decreases production of oxaloacetate. A deficiency in PCC decreases production of succinyl-CoA and causes propionyl-CoA to accumulate, which interacts via a side reaction with oxaloacetate to form methylcitrate. Additionally, low activity of MCC causes methylcrotonyl-CoA to accumulate in the mitochondria where it reacts with glycine ( 9 ) and potentially depletes this amino acid from the mitochondrial matrix. Succinyl-CoA from the TCA cycle and glycine are the precursors for heme biosynthesis. Heme synthesis starts in the mitochondria by condensing succinyl-CoA with glycine to form -aminolevulinate, the first metabolite committed to heme synthesis ( 10 ). We hypothesized that metabolic conditions that interfere with the optimal activity of the TCA cycle may decrease heme synthesis ( 11 ). The rationale for our hypothesis is that all the metabolites produced from the TCA cycle intermediates except heme return to the TCA pool of intermediates during catabolism and/or can be supplied from the diet (e.g. amino acids) ( 8 , 11 ). Heme, however, must be synthesized in situ as dietary heme is degraded and does not return to the TCA cycle intermediate pool after catabolism by heme oxygenase ( 11 ). Therefore, we proposed that when the activity of the TCA cycle is limited, e.g. in deficiency of biotin, the metabolic burden falls mainly on heme synthesis ( 11 ).    Materials and Methods TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED       Materials. Fetal bovine serum (FBS) and FBS stripped by charcoal/dextran (cdFBS) were from Hyclone. PBS, trypsin-EDTA, DMEM, and HEPES (1 mol/L) were from Gibco/Invitrogen. EDTA, Tween-20, biotin, deferoxamine, butyl acetate, and protease inhibitor cocktail were from Sigma-Aldrich. Avidin and HRP-conjugated NeutroAvidin were from Pierce. Iron isotope, as 59FeCl3 (1 Ci/L), was from Perkin Elmer, protein quantitation kit was from Bio-Rad, and liquid scintillation CytoScint was from MP Biomedicals (formerly ICN Biomedicals). Hemin was purchased from Frontier Scientific. Low melting point agarose (LMPA) was NuSieve GTG Agarose from BioWhittaker Molecular Applications and CometSlide was from Trevigen.      In vitro model for BD. cdFBS contains 0.29 µg/L biotin, 0.6% that of normal serum (FBS; 47 µg/L) ( 12 ). We confirmed the low biotin content of cdFBS by using ELISA (data not shown). Compared with the marked change in biotin, the other vitamins, hormones, and minerals were unaffected or minimally affected upon treating with charcoal/dextran ( 12 ). FBS is the only source for biotin in the medium, because biotin, unlike the other micronutrients, is not supplemented in DMEM. Thus, any minor reductions to micronutrient levels other than biotin were replenished by adding DMEM. We demonstrated that cdFBS-supplemented medium causes BD. Establishing this model allowed us to perform long-term experiments and study the consequences of BD on the mitochondria and cellular senescence. Avidin-conjugated beads from Sigma-Aldrich were also used to create BD ( 13 ).      Detection of BDC and specific mitochondrial proteins. The biotinylation status of PC, PCC, and MCC was determined by the avidin blotting technique ( 14 ). The cells were harvested by trypsinization and washed twice with the respective medium. Lysate was prepared in 1% Tween 20, antiproteases, and sonicated in ice. Cellular proteins (100 µg) were resolved in 15% SDS-PAGE, transferred to polyvinylidene difluoride membrane, incubated for 1 h with 1.5 µg in 15 mL PBS NeutrAvidin-HRP conjugate, and detected by chemiluminescence and exposure to imaging film. The different components of the mitochondrial electron transport complexes were evaluated by 15% SDS-PAGE and Western blotting using antibodies against selected subunits from complexes I, III, and IV. For complex IV we used subunit II (COX-II), for complex III we used subunit CorI, and for complex I we used ND39. Quantification of the protein bands in the avidin blot or western blot was performed by densitometry analysis of bands detected on the film using ImageJ software (NIH).      In vitro cellular senescence. Primary human lung fibroblasts (IMR90 from Coriell Institute for Medical Research) are an in vitro model for cellular senescence ( 15 ). IMR90 are started in culture as young cells with a low population doubling level (PDL) and allowed to increase in PDL until senescence (high PDL). At senescence, the cells are viable and metabolically active, although they have lost replicative capacity. To test the effect of BD on PDL, the same batch of IMR90 cells were seeded at 0.5 x 106 per 100-mm dish in: 1) medium supplemented with 10% cdFBS (BD); 2) medium supplemented with 10% normal FBS [biotin-sufficient (BS)]; 3) medium supplemented with 10% cdFBS + 5 µg/L biotin [BD + biotin (BD+B)]; and 4) medium supplemented with 10% normal FBS +5 µg/L biotin [BS + biotin (BS+B)]. All the cultures were split after 7 d, and PDL was calculated as log2 (D/Do), where D and Do are defined as the density of cells at the time of harvesting and seeding, respectively ( 16 ). The cells were seeded again in fresh medium as described above. Additional cells from each splitting cycle were collected and stored at –20°C for further analysis.      Extraction of heme for HPLC analysis. Heme was measured using the HPLC column Bond-Clone-C18, 300 x 3.9 mm (Phenomenex), as previously described ( 17 ). About 5 million cells lysed into 200 µL ice-cold PBS/1 mmol/L EDTA/ 0.2% Tween 20/protease inhibitors. A total of 20 µL of the lysate was used for heme extraction ( 17 ). Heme content was normalized to total protein content (Bio-Rad protein quantification kit).      Measurement of heme synthesis in BD and BS cells. The effect of BD on heme synthesis was tested using the same batch of cells. They were grown on BD, BS, or BD + B (5 or 50 µg/L) media. Heme synthesis was measured using 59Fe, which is incorporated to form heme as described previously ( 18 ). The radioactivity incorporated into newly synthesized heme was measured using a liquid scintillator radiation counter. Total protein content in the sample was measured by Bio-Rad protein assay kit and used for normalization of heme synthesis and iron uptake.      Measuring production of oxidants. 2', 7'-dichlorodihydrofluorescein (DCFH) was used to assay the production of oxidants in cells. IMR90 cells deficient, sufficient, and deficient supplemented for biotin for 3 wk were seeded in 6-well plates with the respective media for 1 wk. The media were removed from the wells and the cells were rinsed with 3 mL Dulbecco's PBS (DPBS). A stock solution of 5 mmol/L DCFH was prepared in ethanol and kept from light. DCFH was added to cells at 25 µmol/L in 3 mL DMEM, incubated at 37°C for 30 min, and followed by 3 washes with 3 mL Hank's DPBS supplemented with magnesium and calcium. Three milliliters of HBSS was added to each well after the final wash. Fluorescence was measured with a CytoFluor 2350 Fluorescent Measuring System plate reader, using 480-nm excitation and emission at 530 nm. For each of the conditions of the cultured cells, a background reading was taken with the procedure described above, omitting the addition of DCFH and including the addition of ethanol. The background reading was subtracted from the actual reading and the fluorescence was normalized to cell number. The cells in each well were counted using a Coulter Counter (Beckman Coulter) after rinsing with DPBS and incubating with trypsin. Results are presented as arbitrary fluorescent units per million cells.      Measuring oxidative damage to DNA by the comet assay. The alkaline (pH >13) comet was performed as previously described ( 19 ). IMR90 cells were grown for 2–4 wk then rinsed, trypsinized, and added to 0.6% LMPA at a concentration of 50,000 cells/0.5 mL LMPA. A total of 50 µL of the cell solution was then added to each CometSlide. After lysing, slides were incubated in alkaline electrophoresis solution followed by electrophoresis at 0.74 V/cm for 20 min, not exceeding 300 mA. Following neutralization with 0.4 mol/L Tris, pH 7.5, and dehydration with ethanol and methanol, slides were incubated for 30 min in SYBR Green diluted 1:10000 in TE (0.01 mol/L Tris + 0.001 mol/L EDTA, pH 7.4) buffer. Slides were then viewed with a fluorescent microscope (Zeiss Axiovert 25 Light/Fluorescence Inverted Microscope) and multiple images of cells were captured (Spot Junior Digital Camera). Individual cells were analyzed using CometScore v1.5 (TriTek). The total number of cells examined was >70 cells per treatment. During analysis, the operator was blind for the identity of the samples.      Statistical analysis. Statistical analyses (t tests, nonparametric Mann-Whitney tests, or 1-way ANOVA) were performed using Prism 4.0 (GraphPad) software. When appropriate, post hoc Tukey, Bonferroni, or Dunn's tests were conducted. Differences were considered significant at P < 0.05. Values in the text are means ± SE.    Results TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED       BD cdFBS medium. Cells grown in cdFBS-supplemented medium lost their BDC ( Fig. 1 ), indicating that cdFBS-supplemented medium was BD. Three BDC, PC, PCC, and MCC, were undetectable in cells maintained for 1 to 2 wk in cdFBS-supplemented medium ( Fig. 1 ). PCC and MCC appeared in the avidin blot as a single wide band, because their molecular mass are similar: 76 and 77 kDa, respectively ( 14 , 20 ). Cells grown in biotin-replenished cdFBS medium showed substantial recovery of BDC ( Fig. 1 ), adding further support that cdFBS-supplemented medium is BD. Based on the results in Figure 1 and additional results presented below, we refer to cells maintained in cdFBS-supplemented medium as BD and cells maintained in medium supplemented with FBS as BS. Similar results were obtained when cells were maintained in serum stripped of biotin using avidin-conjugated beads (data not shown). View larger version (44K): [in this window] [in a new window]   Figure 1  BDC are not detectable in IMR90 cells maintained in BS, BD, BS+B, and BD+B media for 3 wk. PCC and MCC differ by 1 kDa and appear as a single broad protein band. Shown is 1 representative experiment of 6.        Cellular senescence. IMR90 cells had fewer cumulative numbers of PDL until cell senescence when maintained in BD medium, compared with BS or BS+B medium, indicating an increased rate of cellular senescence ( Fig. 2A ). The cells grown in BS medium had a 10.6 gain in PDL compared with cells grown in BD medium ( Fig. 2A ). When the cells were grown in BD+B medium, the cellular senescence did not differ from cells grown in BS medium ( Fig. 2B ), evidence of a cause-and-effect relation between BD and early senescence. Whereas the decrease in BDC started as early as the first week after cells were placed in a BD medium, the effects of BD on PDL were not detectable during the first 2 to 3 wk of the experiment ( Fig. 2B ). This observation suggests that the consequence of BD occurs slowly and cumulatively. Furthermore, an intracellular mechanism may exist that enables young cells to resist the consequences of BD more than old cells, possibly by compensatory upregulation of important, metabolic, salvage, and repair mechanisms. View larger version (15K): [in this window] [in a new window]   Figure 2  Senescence of IMR90 cells maintained in BS, BD, BS+B, and BD+B media. Senescence of IMR90 was determined by calculating the weekly gain in PDL. The cumulative gain in PDL of IMR90 cells from the start until the end of the 16- to 20-wk experiments (A). Values are means ± SE, n = 6. Means without a common letter differ, P < 0.05 (ANOVA, Tukey's test). One representative experiment for IMR90 senescence of 6 shown in A (B). Young cells (low PDL) were split every week, the PDL calculated, and a portion of the cells reseeded. This procedure was repeated until the cells senesced (i.e. no increase in PDL).        Cellular heme content. A 36% decrease in the heme level occurred in cells grown in BD for 2–3 wk (from 38.2 ± 2.4 ng/mg protein in BS to 24.1 ± 1.7 ng/mg protein in BD, P = 0.005, n = 3). A 50% decrease in heme occurred in cells grown in BD for 6 wk (from 27.6 ± 9 ng/mg protein in BS to 12.0 ± 8.5 ng/mg protein in BD, P = 0.03, n = 6). Adding biotin back to the medium restored the heme content to 24.5 ± 3.9 ng/mg protein. The difference in heme levels between the 2 control cell culture likely was due to the different batches of cells used.      Cellular heme synthesis. The rate of heme synthesis in IMR90 cells maintained in BS medium was higher than IMR90 cells maintained in BD medium by 5.21 ng heme mg protein–1 · 3 h–1 (n = 10, Fig. 3A ). Cells maintained in BD medium supplemented with 5 or 50 µg/L biotin synthesized heme at 77 and 93% of the capacity of BS cells, respectively ( Fig. 3A ). Because a change in heme synthesis requires cellular uptake of iron, the changes in iron uptake paralleled those of heme synthesis ( Fig. 3B ). Iron uptake was restored upon adding biotin back to the growth medium. View larger version (13K): [in this window] [in a new window]   Figure 3  Heme synthesis (A) and iron uptake (B) in IMR90 cells maintained in BS, BD, or BD+B (5 or 50 µg/L) media for 3 wk. Values are means ± SE, n = 8. Means without a common letter differ, P < 0.05 (ANOVA, Tukey's test).        Mitochondrial complex IV. Because the TCA cycle is dependent on biotin availability, we measured the levels of mitochondrial complexes I, III, and IV to assess the status of electron transport chain complexes of the mitochondria in cells following 1–2 wk of BD. BD cells had a lower level of complex IV than BS cells, whereas mitochondrial complexes I and III did not differ ( Fig. 4 ). View larger version (53K): [in this window] [in a new window]   Figure 4  Levels of mitochondrial complex IV in IMR90 cells maintained in BS or BD medium for 1–2 wk. Proteins were separated by 15% SDS-PAGE and Western blotted to a polyvinylidene difluoride membrane. The membrane was incubated with antibodies specific for complexes IV (COX-II), I (ND39), and III (CorI). A representative gel is shown.   We compared the kinetics of complex IV's decline after inducing BD in old and young cells. Exposure to short periods of BD (24–72 h) caused an 50% decline in complex IV in old cells within 24 h (P < 0.02) and 48 h (P < 0.0001) of introducing the deficiency. The decline in complex IV in senescent cells due to BD exceeded that in younger cells ( Fig. 5 ). When the BD persisted, complex IV decreased to almost undetectable levels, as in Figure 4 . View larger version (36K): [in this window] [in a new window]   Figure 5  Differential responses to BD in old (PDL >45) and young (PDL <40) IMR90 cells maintained in BS or BD medium for 24, 48, and 72 h. Quantitative representation of the density of the bands in western blots for complex IV similar to that shown in B (48-h incubation) (A). The band density of COX-II was normalized to actin. Values are means ± SD, n = 3. Asterisks indicate different from PDL <40: **P < 0.02, ***P < 0.0001 (Mann-Whitney).   Like heme-a, copper is a structural component in complex IV. Copper is 57% lower in cdFBS and is not included in DMEM. To exclude the possibility that copper contributes to the consequences of BD, we tested whether restoring copper to levels in FBS affects the results of BD. Adding copper did not affect heme synthesis or cellular senescence, suggesting the amount of copper in the cdFBS is enough to sustain normal metabolism of the cells. However, as expected from copper's role in complex IV, the recovery of complex IV was more efficient when copper and biotin were both added to the medium (data not shown).      Oxidant production and DNA damage. The rate of DCFH oxidation in BD cells was twice as high as in BS cells, indicating an increase in the production of oxidants ( Fig. 6A ). Adding biotin back to the growth medium decreased the rate of oxidant production to that of BS cells. DNA damage in BD cells, measured by comet tail moment, was significantly higher than in BS cells, indicating an increase in oxidation of DNA ( Fig. 6B ). Returning biotin to the growth medium decreased the rate of oxidative damage to DNA to the rate in BS cells. View larger version (14K): [in this window] [in a new window]   Figure 6  Oxidants produced (A) and DNA fragmentation (B) in IMR90 cells maintained in BS, BD, BS+B, or BD+B media for 3 wk (A) or 2–4 wk (B). A: Values are means ± SE, n = 6. Means without common letter differ, P < 0.05 (ANOVA, Bonferroni test). B: Values are means ± SE, n = 6. Means without common letter differ, P < 0.05 (ANOVA, Dunn's test).      Discussion TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED   This study established a key role for biotin in maintaining mitochondrial complex IV, heme metabolism, and preventing oxidative damage to DNA in human primary fibroblasts. The metabolic consequences of BD were prevented by returning biotin to the growth medium. The mechanism responsible for these effects of BD likely involves biotin's role in the TCA cycle. The concentration of biotin in BD medium is 100 pmol/L, and in human plasma, 250 pmol/L; how these correspond needs to be studied. BD accelerates cellular senescence in vitro ( 21 ) ( Fig 2 ). Mitochondria have been proposed to play a key role in cellular senescence and aging ( 11 , 22 ). Therefore, key biochemical variables of mitochondria were assayed. BD caused the loss of mitochondrial complex IV but did not affect the mitochondrial complexes I and III ( Fig. 4 ). Selective loss of complex IV increases the ratio of complexes I and III to complex IV and also increases the cellular oxygen concentration. Complex IV is the main oxygen-metabolizing enzyme; it converts >95% of cellular oxygen to water. Deficiency of complex IV is known to increase the production of oxidants by mitochondria ( 23 ). Consistent with this, we found that the levels of oxidants and oxidative damage to DNA were greater in BD cells compared with BS cells ( Fig. 6 ); these alterations may contribute to the acceleration of cellular senescence by BD ( Fig. 2 ). We have previously demonstrated that the antioxidant N-tert-butyl hydroxylamine prevents cdFBS-induced early senescence ( 21 ). Heme level and synthesis were markedly decreased in BD cells ( Fig. 3 ), indicating that adequate heme synthesis requires biotin and that BD can cause heme deficiency. Thus, biotin should be considered the 8th member of the group of vitamins and minerals required for adequate heme synthesis ( 11 ). The decrease in iron uptake in BD cells ( Fig. 3 ) is unexpected, because heme deficiency should be expected to cause a compensatory increase in iron uptake ( 24 , 25 ). A possible explanation for the lack of an increase in iron uptake in BD cells is that the heme deficiency caused by BD is due to a decrease in succinyl-CoA, which lowers the production of porphyrins. Porphyrins are intermediates in the biosynthesis of heme. These results suggest that optimal uptake of iron requires that the mechanisms for iron assimilation into heme remain intact. Adequate levels of biotin appear to be essential for adequate iron uptake. Thus, for correcting iron deficiency in humans, it may be important to ensure biotin adequacy. Heme is essential in intermediary metabolism, gene regulation, and mitochondrial integrity ( 26 ). In this study, we used the decrease in complex IV in response to BD as a functional indicator of heme deficiency ( Fig. 4 ). We previously demonstrated that heme deficiency causes selective inactivation of mitochondrial complex IV ( 27 , 28 ). A likely reason for this effect is that heme is a precursor for heme-a. Heme-a is a unique type that exists only in complex IV. The maturation of heme to heme-a requires important biochemical modifications ( 29 ). Thus, a shortage in heme causes a decrease in heme-a, leading to a selective decrease in mitochondrial complex IV. Complex IV is an enzyme complex made of 13 subunits (e.g. subunits COX-I, COX-II, COX-III, etc.) ( 30 ). The assembly of complex IV starts with the assimilation of heme-a into COX-I, followed by assembly of COX-IV&II, then the rest of the subunits. Thus, the assembly of the entire complex depends on proper folding of COX-I ( 31 , 32 ), which becomes limiting in heme deficiency. We used subunit COX-II as representative of the entire complex ( 28 ). With the exception of complex IV, heme proteins, such as complex III, use heme directly and have no need for maturation. A selective decline in complex IV triggers the production of oxidants and free radicals by the mitochondria ( 27 , 28 ). Therefore, it is likely that heme deficiency is the mechanism by which BD causes selective loss of complex IV, mitochondrial decay, and oxidative stress and accelerates cellular senescence. A limitation in the supply of TCA cycle intermediates may also decrease the production of ATP and limit the utilization of energy from biochemical sources. This change in energy metabolism may also contribute to the metabolic consequences of heme deficiency. We, and others, have demonstrated that low levels of heme do not immediately decrease cell viability, but rather low levels disrupt cellular responses to stress, block differentiation, and alter intermediary metabolism ( 27 , 33 , 34 ). Old cells are more susceptible to BD than young cells ( Fig. 5 ). This finding is consistent with old cells being slower than young cells to recover from heme deficiency ( 28 ). We therefore propose that mitochondria from old tissues are more susceptible to BD and heme deficiency ( 28 ). An age-dependent elevation in biotin absorption has been shown in old rats ( 7 ). In humans, plasma biotin concentrations appear to increase with age ( 35 ). Because plasma biotin does not always reflect tissue biotin ( 36 ), this increase may indicate a change in biotin metabolism with age. More research is needed to determine the relevance of these findings to the biology of aging in humans. The mechanistic links among biotin, the TCA cycle, the integrity of heme metabolism, and mitochondria may provide in part an explanation for the teratogenic consequences of BD in rodents ( 2 ). The requirement of biotin for the metabolic integrity of the developing fetus appears high ( 4 ), probably due to rapid growth and increase in mass. Recent studies showed that proliferation of lymphocytes consumes large amounts of biotin ( 37 ), which might also be true for other types of cells. Therefore, the teratogenic consequences of reduced availability of biotin may be triggered in part by reduced mitochondrial function, abnormal heme metabolism, and abnormal histone biotinylation. All of these metabolic activities are likely to be critical for normal development. The effect of biotin on glucose status in diabetes mellitus patients may be a result of improved mitochondrial function, particularly the TCA cycle ( 38 , 39 ). The TCA cycle may also fail to supply enough succinyl-CoA when there are deficiencies of the vitamins that are essential for the production of TCA cycle intermediates. A combination of deficiencies and exposure to toxins that require increased heme synthesis would aggravate the cell's need for succinyl-CoA ( 11 ). The importance of biotin in heme synthesis and maintaining the TCA cycle warrants further investigation to the extent of BD as well as the optimal levels of biotin needed by the population.    ACKNOWLEDGMENTS   The authors are grateful to K. Boyle, J. McCann, J. Nides, and F. Viteri for commenting on the manuscript.    FOOTNOTES   1 Supported by the Bruce and Giovanna Ames Foundation (H.A.); the National Center for Minority Health and Health Disparities Center Grant P60 MD00222 (B.N.A., H.A.); National Foundation of Cancer Research Grant M2661 (B.N.A.); and National Center for Complementary and Alternative Medicine K05 AT001323 (B.N.A.). 2 Abbreviations used: ACC, acetyl-CoA carboxylase; BD, biotin deficient/deficiency; BDC, biotin-dependent carboxylase; BD+B, BD + biotin; BS, biotin-sufficient/sufficiency; BS+B, BS + biotin; cdFBS, FBS stripped by charcoal/dextran; FBS, fetal bovine serum; MCC, 3-methylcrotonyl-CoA carboxylase; PDL, population doubling level; PCC, propionyl-CoA carboxylase; PC, pyruvate carboxylase; TCA, tricarboxylic acid (Krebs) cycle; LMPA, low melting point agarose; DPBS, Dulbecco's PBS; DCFH, 2', 7'-dichlorodihydrofluorescein. Manuscript received 10 July 2006. Initial review completed 17 August 2006. Revision accepted 2 November 2006.    LITERATURE CITED TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED   1. Said HM. Biotin: the forgotten vitamin. Am J Clin Nutr. 2002;75:179–80. [Free Full Text] 2. Mock DM. Marginal biotin deficiency is teratogenic in mice and perhaps humans: a review of biotin deficiency during human pregnancy and effects of biotin deficiency on gene expression and enzyme activities in mouse dam and fetus. J Nutr Biochem. 2005;16:435–7. [Medline] 3. Watanabe T, Endo A. Teratogenic effects of maternal biotin deficiency on mouse embryos examined at midgestation. Teratology. 1990;42:295–300. [Medline] 4. Mantagos S, Malamitsi-Puchner A, Antsaklis A, Livaniou E, Evangelatos G, Ithakissios DS. Biotin plasma levels of the human fetus. Biol Neonate. 1998;74:72–4. [Medline] 5. Mock DM. Biotin. 7th ed. Washington: International Life Sciences Institute Press; 1996. 6. Dakshinamurti K, Sabir MA, Bhuvaneswaran C. Oxidative phosphorylation by biotin-deficient rat liver mitochondria. Arch Biochem Biophys. 1970;137:30–7. [Medline] 7. Said HM, Horne DW, Mock DM. Effect of aging on intestinal biotin transport in the rat. Exp Gerontol. 1990;25:67–73. [Medline] 8. Owen OE, Kalhan SC, Hanson RW. The key role of anaplerosis and cataplerosis for citric acid cycle function. J Biol Chem. 2002;277:30409–12. [Free Full Text] 9. Bartlett K, Ng H, Leonard JV. A combined defect of three mitochondrial carboxylases presenting as biotin-responsive 3-methylcrotonyl glycinuria and 3-hydroxyisovaleric aciduria. Clin Chim Acta. 1980;100:183–6. [Medline] 10. Maines MD, Kappas A. Metals as regulators of heme metabolism. Science. 1977;198:1215–21. [Abstract/Free Full Text] 11. Atamna H. Heme, iron, and the mitochondrial decay of ageing. Ageing Res Rev. 2004;3:303–18. [Medline] 12. Wilkinson RF. The effect of charcoal/dextran treatment on select serum components. In: Art to science in tissue culture (Hyclone). Vol 12:1. Logan (UT): Hyclone Laboratories; 1993. 13. Rodriguez-Pombo P, Sweetman L, Ugarte M. Primary cultures of astrocytes from rat as a model for biotin deficiency in nervous tissue. Mol Chem Neuropathol. 1992;16:33–44. [Medline] 14. Thampy KG. Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of A carboxylase from rat heart. J Biol Chem. 1989;264:17631–4. [Abstract/Free Full Text] 15. Juckett DA. Cellular aging (the Hayflick limit) and species longevity: a unification model based on clonal succession. Mech Ageing Dev. 1987;38:49–71. [Medline] 16. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci USA. 1994;91:4130–4. [Abstract/Free Full Text] 17. Atamna H, Frey WH II. A role for heme in Alzheimer's disease: heme binds amyloid beta and has altered metabolism. Proc Natl Acad Sci USA. 2004;101:11153–8. [Abstract/Free Full Text] 18. Atamna H, Boyle K. Amyloid-beta peptide binds with heme to form a peroxidase: relationship to the cytopathologies of Alzheimer's disease. Proc Natl Acad Sci USA. 2006;103:3381–6. [Abstract/Free Full Text] 19. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35:206–21. [Medline] 20. Rodriguez-Melendez R, Perez-Andrade ME, Diaz A, Deolarte A, Camacho-Arroyo I, Ciceron I, Ibarra I, Velazquez A. Differential effects
  • June 26, 2012 The oral administration of the pyrimidine precursor orotic acid in doses of 3 to 6 Gm. daily to patients with pernicious anemia in relapse produced with some regularity Partial remissions in the manifestations of vitamin B 12 deficiency. The best response occurred in a patient with postgastrectomy pernicious anemia. Little effect was seen in two undernourished patients who responded well, nevertheless, to B 12 therapy. Irregularity in the absorption of this poorly soluble compound from the intestinal tract, possible changes en route, and other limiting nutritional factors may account for some variation in response. Preparations suitable for parenteral administration have not been developed. The early effects of orotic acid in pernicious anemia resembled those of small amounts of B 12 . Reticulocytosis appeared 7 to 14 days after the start of therapy. Gradual clinical and hematologic improvement followed. We have not been able to evaluate the possible ability of this compound to reverse the neurologic manifestations of pernicious anemia. Complete remissions in the disease were never produced, however, by orotic acid, for at the height of improvement, red cell macrocytosis and some degree of megaloblastic cellular development persisted in the bone marrow. Patients maintained on orotic acid alone for 5 to 7 months gradually relapsed with increasing anemia and lingual mucosal atrophy. Toxic effects of the chemical were not seen. 10 In no instance did it produce an effect in pernicious anemia like that of folic acid: a quick but suboptimal response at first, followed ultimately by relapse, with neurologic disease, lingual mucosal atrophy, and/or anemia with a hypocellular nonmegaloblastic bone marrow—all comparatively refractory at this stage to vitamin B 12 therapy. Since orotic acid is known to function only as an intermediate in the synthesis of pyrimidines 2, 9 (fig. 12), the hematopoietic effects of some precursors and derivatives of the compound were studied. None proved to be as active as orotic acid. Carbamyl aspartic acid given in doses of 3 Gm. daily was followed by a slight reticulocytosis in two patients. Aspartic acid given in doses up to 15 to 20 Gm./day with 3 to 6 Gm. of orotic acid had little or no effect in two patients. A concentrate of uridylic and cytidylic acids obtained from yeast, probably absorbed from the intestinal tract as nucleosides, showed some effect in one of the two patients to whom it was given. This same preparation was remarkably effective when given to a child with a congenital abnormality in pyrimidine biosynthesis who excreted large amounts of orotic acid in the urine. 13 The parenteral administration of thymidine, 0.5 Gm. daily for six days, had no hematopoietic effect in one patient, but a significant response occurred when inosine was given with it concurrently. Sodium deoxyribonucleic acid (DNA) prepared from fish sperm by the hot alkaline extraction method produced a moderate reticulocytosis in one patient. A second crest followed the addition of orotic acid to her regimen. A less hydrolyzed DNA preparation given to another patient for one week had no effect, but there was apparently none either when orotic acid was given 6 Gm./day for the same length of time. Vitamin B 12 and folic acid appear to have similar, overlapping or reciprocal, actions in different biologic systems. A comparison of the hematologic effects of folic acid metabolites with that of orotic acid was accordingly undertaken. Methionine reduces the vitamin B 12 requirement of bacteria, and the de novo synthesis of this amino acid is increased by cobalamin. 14-16 Methionine given to patients with pernicious anemia in relapse, however, seemed to depress hematopoiesis. It is of interest that the methyl group of methionine is not utilized for the biosynthesis of thymine in bacteria. The methyl group of thymine was derived from glucose instead. 17 The hematopoietic effect of serine has not yet been studied in patients with pernicious anemia in relapse. Histidine, which is synthesized by folic acid containing enzymes, was given with the thought that it might become an "essential" amino acid under some circumstances, or provide a general source of active formate or of transferable formimino groups usable in protein and/or nucleic acid synthesis, 16,18 A definite hematopoietic stimulus was obtained from DL-histidine in two Patients with pernicious anemia in relapse. The response was quick and suboptimal, however, and did not potentiate that of orotic acid. One patient in partial hematologic remission after taking histidine for 12 weeks was then given folic acid. Additional benefit was not observed. Further studies of the effect of these and other folic acid metabolites in pernicious anemia and in nutritional megaloblastic anemia are in progress. Biochemical studies in the past have given no indication that vitamin B 12 is concerned with pyrimidine biosynthesis. The latter process, of considerable current interest also in reference to the development of pyrimidine antimetabolites, is outlined in figure 12. 19-23 Carbamyl phosphate and aspartic acid are converted by known enzymatic reactions to orotic acid. The latter is rapidly converted to pyrimidine nucleotides, derivatives, and to the pyrimidine moieties of nucleic acids. Hurlbert and Potter injected small amounts of orotic acid intraperitoneally into rats. 19 About one-third of the chemical was immediately excreted unchanged and another third immediately taken up by the liver. Orotic acid was quickly converted in the liver quantitatively to acid-soluble metabolites, particularly uridine-5'-phosphate, derivatives of this compound, and cytidine-5'-phosphate. The uridine phosphate pool appeared to be the immediate metabolic precursor of the uracil of the ribonucleic acid (RNA) in the nucleus and a major source of the pyrimidines of the cytoplasmic RNA. A large contribution to liver DNA pyrimidine was observed in tissue regenerating after partial hepatectomy. 24 Thymidine is formed in vitro from uracil deoxyriboside by a reaction inhibited by Aminopterin. 25 Vitamin B 12 may facilitate nucleoside and nucleic acid synthesis by different mechanisms in different biologic systems. In bacteria there is evidence that it promotes the synthesis of methionine, nucleosides and/or deoxyribose, and possibly activates protein sulfhydryl groups. 14 In animals it may promote methyl group neogenesis, but there is increasing doubt that it has anything to do with transmethylation. 16,26 The indications are that vitamin B 12 has different function(s) than that of folic acid, which is concerned in the synthesis, transfer and/or incorporation of formate, formimino and hydroxymethyl groups. The vitamin B 12 requirement of the human adult on a weight basis is 10 to 15 times less than that of animals, 26 but a disease unique to man, pernicious anemia, results from its lack. Evidence relating the deficiency in pernicious anemia to pyrimidines was first obtained by Vilter and associates who reported incomplete hematologic remissions in patients to whom they gave 15 to 30 Gm. of thymine or uracil daily. 11, 12 Thymine was effective in two of their patients who had relapsed while taking folic acid. Uracil had no effect in a patient with megaloblastic anemia of pregnancy, but who did respond to thymine. Nieweg, et al., studied the relationship of vitamin B 12 to folic acid in the megaloblastic anemias. They cited evidence to support the idea that in the human, vitamin B 12 was particularly concerned with pyrimidine formation and RNA-protein synthesis. 27 The degree of remission that can be produced in patients with pernicious anemia in relapse by the administration of orotic acid suggests, too, that one major consequence of vitamin B 12 deficiency in the human is a defect in pyrimidine biosynthesis and/or incorporation. Other processes, such as purine ring formation, may also be affected. The mechanism by which orotic acid induces partial remissions in pernicious anemia is unknown. It could serve merely as a metabolite which when supplied from exogenous sources would circumvent a block in its synthesis or in that of a precursor. Increasing the supply of orotic acid could possibly overcome by mass action a defect in the synthetic pathway at a later stage. In view of demonstrated feed-back regulatory mechanisms in pyrimidine synthesis, 28 however, there are too many ways by which orotic acid could influence metabolism in the presence of vitamin B 12 deficiency to justify further speculation.
  • June 26, 2012 TO THE EDITOR: Anemia is not usually mentioned as a complication of vitamin D intoxication but has been described in patients with and without renal failure [1] . We report on a woman with vitamin D intoxication and anemia not caused by renal failure. A 66-year-old woman was admitted to the emergency department with hypercalcemia diagnosed after 3 weeks of severe constitutional symptoms. Three years earlier, osteoporosis had been diagnosed and a rheumatologist had prescribed an extemporaneous formulation (200 IU of vitamin D and 1 g of calcium glucobionate twice daily), which was prepared by a pharmacist. Blood tests at admission showed a calcium level of 4.04 mmol/L, a hemoglobin concentration of 103 g/L, a urea concentration of 11.2 mmol/L, and a creatinine level of 146 µmol/L. After rehydration, the hemoglobin concentration decreased to 83 g/L. Anemia was nonspecific and nonregenerative, and results of additional tests (chest radiography, mammography, abdominal ultrasonography, bone scintigraphy, fibrogastroscopy, and colonoscopy) were normal. Parathyroid hormone was undetectable, and the plasma 25-hydroxyvitamin D level was 696 nmol/L (normal range, 15 to 125 nmol/L). Two months later, while the patient was receiving a milk-free diet, plasma 25-hydroxyvitamin D levels were high, serum calcium levels were normal, and anemia had resolved. Symptoms had begun roughly when a new bottle of pills with a vitamin D content of 200 µg (8000 IU) was started. The association of hypercalcemia and anemia suggested a neoplastic origin; this idea was rejected when results of additional examinations became available. High vitamin D levels could directly affect hematopoietic cells [2] or act through high calcium levels, which inhibit erythroid colony formation in vitro [3] and erythropoietin production in vitro [4] and in vivo [5] . That calcium is more important than vitamin D itself is supported by the course of our patient, whose anemia subsided after normalization of calcium levels, despite high vitamin D levels. In addition to the danger of extemporaneous formulations, which carry a higher risk for error than factory-made pills, anemia is another potential complication of vitamin D intoxication. 1. Scharfman WB, Proop S. Anemia associated with vitamin D intoxication. N Engl J Med. 1956; 255:1207-12. 2. Reichel H, Koeffler HP, Norman AW. Production of 1a,25-dihydroxyvitamin D3 by hematopoietic cells. Prog Clin Biol Res. 1990; 332:81-97. 3. Misiti J, Spivak JL. Erythropoiesis in vitro. J Clin Invest. 1979; 64:1573-9. 4. Nagakura K, Ueno M, Brookins J, Beckman BS, Fisher JW. Effects of low calcium levels on erythropoietin production by human renal carcinoma cells in culture. Am J Physiol. 1987; 253:797-801. 5. McGonigle RJ, Brookins J, Pegram BL, Fisher JW. Enhanced erythropoietin production by calcium entry blockers in rats exposed to hypoxia. J Pharmacol Exp Ther. 1987; 241:428-32.
  • Transcript

    1. Isfahan University of Medical Science, School of Vitamins & Cofactors Anemia & Vitamins Pharmacy Department of Clinical BiochemistryJune 26, 2012 Total slides : 120 1
    2. A n e m ia & Vit aminsBy:A.N. Emami Razavi
    3. Vitamins & Cofactors Anemia & Vitamins Which vitamin deficiency do you think involved in anemia? Which vitamin deficiency do you think not involved in anemia?June 26, 2012 Total slides : 120 3
    4. Vitamins & Cofactors Anemia & Vitamins O u t lin e s  A n e m ia  In t r o d u c t io n t o v it a m in s a n d a n e m ia  T h ia m in & a n e m ia  R ib o f la v in & a n e m ia  N ia c in & a n e m ia  P a n t o t h e n ic a c id & a n e m ia  P y r id o x in e & a n e m ia  C o b a la m in & a n e m ia  F o lic a c id & a n e m ia  V it a m in A & a n e m ia  V it a m in K & a n e m ia  V it a m in E & a n e m ia  V it a m in C & a n e m ia  B io t in & a n e m ia  O r o t ic a c id & a n e m ia  O t h e r v it a m in s & a n e m iaJune 26, 2012 Total slides : 120 4
    5. AnemiaAnemia & Vitamins
    6. Vitamins & Cofactors Anemia & Vitamins Definition of Anemia  A deficiency in the size or number of red blood cells or in the amount of hemoglobin a red blood cell contains  Decrease in blood hemoglobin below a person’s physiological need  Hemoglobin concentration below 95th percentile of healthy reference populationJune 26, 2012 Total slides : 120 6
    7. Vitamins & Cofactors Anemia & Vitamins Causes of Anemia  Lack of required nutrients  Loss of blood  Chronic Disease  Genetic Abnormalities  Inadequate production of red blood cellsJune 26, 2012 Total slides : 120 7
    8. Vitamins & Cofactors Anemia & Vitamins Signs and symptoms of Anemia Anemia occurs in many types, but the main symptom of most anemias is fatigue. Thats true for vitamin deficiency anemias, which can also result in:  Pale skin  Sore mouth and tongue  Shortness of breath  Loss of appetite  Diarrhea  Numbness or tingling in your hands and feet  Muscle weakness  Mental confusion or forgetfulness  Vitamin deficiencies usually develop slowly, over several months to years. Vitamin deficiency symptoms may be subtle at first, but they increase as the deficiency worsensJune 26, 2012 Total slides : 120 8
    9. Vitamins & Cofactors Anemia & Vitamins Classification of Anemia Based on cell size (MCV)  Macrocytic (large) MCV 100+ fl (femtoliters)  Normocytic (normal) MCV 8-99 fl  Microcytic (small) MCV<80 fl Based on hemoglobin content (MCH)  Hypochromic (pale color)  Normochromic (normal color)June 26, 2012 Total slides : 120 9
    10. Vitamins & Cofactors Anemia & Vitamins Types of Anemia  Anemia due to B12 deficiency  Anemia due to folate deficiency  Anemia due to iron deficiency  Hemolytic anemia  Hemolytic anemia due to G-6-PD deficiency  Idiopathic aplastic anemia  Idiopathic autoimmune hemolytic anemia  Immune hemolytic anemia  Megaloblastic anemia  Pernicious anemia  Secondary aplastic anemia  Sickle cell anemiaJune 26, 2012 Total slides : 120 10
    11. Vitamins & Cofactors Anemia & Vitamins Introduction to vitamins and anemia  Your body needs vitamins ( nutrients found in most foods) for many reasons, including producing healthy red blood cells. If your body is deficient in certain key vitamins, you can develop a type of anemia ( a condition in which your blood is low on healthy red blood cells ) called vitamin deficiency anemia.  Red blood cells carry oxygen from your lungs to all parts of your body. Without enough healthy red blood cells, your body cant get the oxygen it needs to feel energized. To produce red blood cells, your body needs iron and certain vitamins along with adequate protein and calorie intake.  Vitamin deficiency anemia can also lead to other health problems. Fortunately, you can usually correct vitamin deficiency anemia with supplements and dietary changes.June 26, 2012 Total slides : 120 11
    12. Thiamin & Anemia Anemia & Vitamins
    13. Vitamins & Cofactors Anemia & Vitamins TRMA  Thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness is an autosomal recessive disorder; reported in less than 30 families.  Megaloblastic anemia occurs between infancy and adolescence. The anemia is corrected with pharmacologic doses of thiamine (vitamin B1) (25-75 mg/day compared to US RDA of 1.5 mg/day). However, the red cells remain macrocytic. The anemia can recur when thiamine is withdrawn. Progressive sensorineural hearing loss has generally been early and can be detected in toddlers, is irreversible, and may not be prevented by thiamine treatment. The diabetes mellitus is non-type I in nature, with age of onset from infancy to adolescence.June 26, 2012 Total slides : 120 13
    14. Vitamins & Cofactors Anemia & Vitamins Diagnosis/testing  The diagnosis of TRMA is based on an obligate triad of clinical features described above. Examination of the bone marrow reveals megaloblastic anemia with erythroblasts often containing iron-filled mitochondria (ringed sideroblasts).  SLC19A2, which encodes the high-affinity thiamine transporter, is the only gene known to be associated with TRMA. All individuals with the diagnostic phenotypic triad evaluated by sequence analysis have identifiable mutations in the SLC19A2 gene. Sequence analysis of SLC19A2 DNA is available clinically.June 26, 2012 Total slides : 120 14
    15. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 15
    16. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 16
    17. Vitamins & Cofactors Anemia & Vitamins Biochemical mechanism  The underlying biochemical mechanisms responsible for these conspicuous changes are, however, not very well defined and remain somewhat speculative and controversial.  There are basically 2 current theories, both rooted in the concept that nucleotide synthesis is impaired as that in folate and cobalamin (vitamin B12) deficiency.June 26, 2012 Total slides : 120 17
    18. Vitamins & Cofactors Anemia & Vitamins  In one theory, lack of deoxythymidine triphosphate (dTTP) retards the elongation of newly formed replicating segments of DNA, resulting in fatally fractured pieces that trigger premature apoptosis.  In the other theory, build-up of deoxyuridine triphosphate (dUTP) resulting from failure of conversion of dU to thymidine causes an inordinate accumulation of dUTP, which can then substitute for missing dTTP in the machinery of DNA polymerase activity. Mis-incorporation of dUTP results in excision of the faulty segment followed by misrepair while the famine for dTTP persists, and thus ensues a futile cycle of excision-misrepair. This, too, results in apoptosis, the final common pathway of ineffective hematopoiesis in megaloblastic anemia.June 26, 2012 Total slides : 120 18
    19. Vitamins & Cofactors Anemia & Vitamins Role of thiamin in ribose 5-phosphate synthesis  Through tracking the stable 13C-labeled glucose in fibroblasts from patients with TRMA, Boros and colleagues concluded that the underlying lesion in this condition resides in the pentose cycle, specifically the transketolase enzyme, which requires thiamine pyro-phosphate as a cofactor.  Through a consideration of the several interconnected pathways of glycolysis, the tricarboxylic acid cycle, and ribose synthesis, the authors defined substrate flux in TRMA and normal wild-type fibroblasts grown in both low- and high- thiamine medium.June 26, 2012 Total slides : 120 19
    20. Vitamins & Cofactors Anemia & Vitamins  They concluded that defective high-affinity thiamine transport in TRMA leads to a critical reduction in de novo generation of ribose with consequent cell-cycle arrest that triggers precocious apoptosis. Their results clearly demonstrate a selective and time-dependent loss of ribose synthesis in TRMA patients that is most marked under thiamine-deprived culture conditions and is partially restored by thiamine supplementation, explaining the clinical responsiveness of TRMA patients to high doses of thiamine.June 26, 2012 Total slides : 120 20
    21. Vitamins & Cofactors Anemia & Vitamins Thiamin and pyruvate dehydrogenase complexJune 26, 2012 Total slides : 120 21
    22. Vitamins & Cofactors Anemia & Vitamins Role of the TCA cycle in anabolismJune 26, 2012 Total slides : 120 22
    23. Vitamins & Cofactors Anemia & Vitamins Diagnosis  To establish the extent of disease in an individual diagnosed with thiamine-responsive megaloblastic anemia syndrome (TRMA), the following evaluations are recommended:  Peripheral blood count and bone marrow analysis for evidence of megaloblastic anemia  Serum folate concentration, serum vitamin B12 concentration, and serum iron studies to exclude other entities  Fasting serum glucose concentration, oral glucose tolerance test (OGTT), and urine analysis to diagnose diabetes mellitus  Hearing test  Ophthalmologic evaluation  Cardiac evaluation, including echocardiographyJune 26, 2012 Total slides : 120 23
    24. Vitamins & Cofactors Anemia & Vitamins Treatment  Early administration of pharmacologic doses of oral thiamine (25-75 mg/day compared to US RDA of 1.5 mg/day) ameliorates the megaloblastic anemia and the diabetes mellitus. It may prevent further deterioration of hearing function.  Whether treatment with thiamine from birth, or even prenatally, could reduce the hearing defect is a matter of conjecture.June 26, 2012 Total slides : 120 24
    25. Riboflavin & Anemia Anemia & Vitamins
    26. Vitamins & Cofactors Anemia & Vitamins  Riboflavin deficiency has been associated with the development of normochromic ,normocytic anaemia .  It may be one of the most common vitamin deficiencies among the people of developing nations.  This anemia is associated with reticulocytopenia; leukocytes and platelets are generally normal.  Administration of riboflavin to deficient patients causes reticulocytosis, andJune 26, 2012 Total slides : 120 26
    27. Vitamins & Cofactors Anemia & Vitamins Biochemical mechanism  Effects on iron absorption:  A FMN-dependent oxidoreductase (NADPH-ferrihemoprotein reductase) catalyses the removal of iron from storage ferritin (by reducing heme-thiolate-dependent monooxygenases).  Riboflavin affects iron absorption by maintaining the absorptive capacity of gastrointestinal villi .  Effects on heme metabolism  Protoporphyrinogen oxidase at the iner mitochondrial membrain contains one FAD moiety per homodimer ,oxidizes protoporphyrinogen-IX to protoporphyrin-IX.  NADPH dehydrogenase (EC1.6.99.1) reduces biliverdin to bilirubin in the liver and also may protect against oxidative damage.June 26, 2012 Total slides : 120 27
    28. Vitamins & Cofactors Anemia & Vitamins Effects on other vitamins metabolism that their deficiencies are related to anemia:  Metabolism of vitamin B12  Cobalamin reductase  Aquacobalamin reductase/NADPH  Aquacobalamin reductase/NADH  Metabolism of vitamin B6:  Pyridoxamine-phosphate oxidase interconverts the B6 vitamins pyridoxamine,pyridoxine and pyridoxal, as well as their phosphates.  Metabolism of folic acid:  The FAD-dependent methylen tetrahydrofolate reductase is needed for folate metabolite recycling(with a reduction of its activity higher folate intakes are needed to avoid deficiency).June 26, 2012 Total slides : 120 28
    29. Vitamins & Cofactors Anemia & Vitamins  Metabolism of vitamin B2:  Maintains supplies of vitamin B3 with the help of an enzyme kynurenine mono-oxygenase and vitamin B2 in its FAD form.  Metabolism of vitamin C:  Thioredoxin reductase regenerates reduced glutathione, which is used for dehydroascorbate reductase.  Metabolism of vitamin K:  NADPH dehydrogenase (EC1.6.99.6) and two forms of NAD(P)H dehydrogenase (EC1.6.99.2) reactive vitamin K (dicomarol inhibitable) and also provide important antioxidant protection.  Metabolism of vitamin A:  Retinal dehydrogenase is the enzyme that generates retinoic acid from retinal.June 26, 2012 Total slides : 120 29
    30. Niacin & Anemia Anemia & Vitamins
    31. Vitamins & Cofactors Anemia & Vitamins  Niacine deficiency has produced macrocytic anaemia among human patients with pellagra although this is usually due to an accompanying deficiency in folic acid.  Niacin nutritional deficiency causes hemorrhagic diarrhea, dermatitis, anemia and a severe stomatitis with ulceration of the mouth and tongue (black tongue).June 26, 2012 Total slides : 120 31
    32. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 32
    33. Vitamins & Cofactors Anemia & Vitamins Role of niacin in citric acid cycle NAD+ NADH + H+ Isocitrate Alpha-ketogluterate NAD+ NADH + H+ Alpha-ketogluterate Succinyl CoA NAD+ NADH + H+ Malate OxaloacetateJune 26, 2012 Total slides : 120 33
    34. Pantothenate & Anemia Anemia & Vitamins
    35. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 35
    36. Vitamins & Cofactors Anemia & Vitamins  Rats fed a purified diet low in pantothenic acid developed granulocytopenia and anemia singly or in combination. In the former, the marrow showed marked depletion of granulocytes, particularly of the more mature cells, and a slight increase in erythroid cells. In combined granulocytopenia and anemia the granulocytes of the marrow were still further reduced and the erythroid cells were also depleted. Marked reduction in the number of megakaryocytes occurred both in the granulocytopenic and in the granulocytopenic and anemic rats.  Following treatment with combined folic acid, pantothenic acid, and niacinamide, granulocytopenic rats responded by showing a prompt rise in lymphocyte and polymorphonuclear leukocyte count, marked granulocyte response of the bone marrowJune 26, 2012 Total slides : 120 36
    37. Vitamins & Cofactors Anemia & Vitamins Biochemical mechanism  Pantothenic acid as a part of coenzyme A is essential for Heme formation in hemoglobin.  The production of acetyl-C0A from pyruvate and succinyl- CoA from alpha-ketoglutarate constantly consumes large amounts of CoA.  Succinyl-CoA is needed for D-ALA synthesis the first step in heme production.June 26, 2012 Total slides : 120 37
    38. Pyridoxine & Anemia Anemia & Vitamins
    39. Vitamins & Cofactors Anemia & Vitamins  Vitamin B6 (pyridoxine) deficiency can disturb heme synthesis and lead to normocytic, microcytic or sideroblastic anemia. Treatment of sideroblastic anemia with vitamin B6 has resulted in the restored activity of erythroblastic δ-aminolevulinic acid synthetase (ALAS), the rate-limiting enzyme in heme synthesis, followed by correction of the hematological abnormalities.  Heme biosynethsis begins in the mitochondrion with the formation of 5-aminolevulinic acid. This molecule moves to the cytosol where a number of additional enzymatic transformations produce coproporphyrinogin III. The latter enters the mitochondrion where a final enzymatic conversion produces protophorphyrin IX. Ferrochelase inserts iron into the protophorin IX ring to produce heme.June 26, 2012 Total slides : 120 39
    40. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 40
    41. Vitamins & Cofactors Anemia & Vitamins  In Germany, after treating children hospitalized with iron deficiency anemia for 8 days with iron plus vitamin B6, there was an apparent acceleration of heme synthesis, reflected in Hb concentrations that were higher than observed in children who received only ironJune 26, 2012 Total slides : 120 41
    42. Vitamins & Cofactors Anemia & Vitamins  Vitamin B6 may also inhibit sickling of erythrocytes in sickle-cell anemia (SCA), possibly increasing erythrocyte counts, Hb concentrations and Hct among SCA patients  Vitamin B6 deficiency is rare, but treatment with B6 may be effective in correcting the hematological abnormalities of sideroblastic anemia.June 26, 2012 Total slides : 120 42
    43. Vitamins & Cofactors Anemia & Vitamins  The bone marrow aspirate from a patient with sideroblastic anemia in this photomicrograph was stained with Perls Prussian blue. The arrow indicates a normoblast with a greenish halo of material stained by Perls Prussian blue surrounding the nucleus. Electron microscopic examination would should these to be iron-laden mitochondria.June 26, 2012 Total slides : 120 43
    44. Vitamins & Cofactors Anemia & Vitamins Effect of Vitamin B6 on niacin synthesis TRYPTOPHAN N-FORMYLKYNURENINE KYNURENINE Xanthurenic 3-OH-KYNURENINE Acid Kynureninase (PLP) acetyl CoA 3-OH ANTHRANILIC ACID acetoacetyl QUINOLINIC ACID CoA NIACINJune 26, 2012 Total slides : 120 44
    45. Cobalamin & Anemia Anemia & Vitamins
    46. Vitamins & Cofactors Anemia & Vitamins B12 deficiency Anemia This picture shows large, dense, oversized, red blood cells (RBCs) that are seen in megaloblastic anemia. Megaloblastic anemia can occur when there is a deficiency of vitamin B-12. Megaloblastic anemia - view of red blood cellsJune 26, 2012 Total slides : 120 46
    47. Vitamins & Cofactors Anemia & Vitamins CAUSES OF MACROCYTOSIS OTHER MDS LIVER ETOH HEMOL DRUGS B12/ FOLATEJune 26, 2012 Total slides : Total slides : 120 47
    48. Vitamins & Cofactors Anemia & Vitamins Age of onset 35 30 25 20 15 10 5 0 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100June 26, 2012 Total slides : 120 48
    49. Vitamins & Cofactors Anemia & Vitamins Causes  Pernicious anemia  Rare autoimmune disease  Failure to absorb B12 from food  Very common in older patients  Drugs  Metformin  PPI  Dramatic reduction in B12 absorptionJune 26, 2012 Total slides : 120 49
    50. Vitamins & Cofactors Anemia & Vitamins Physiologic roles of vitamin B12  Conversion of propionyl-CoA to methylmalonyl CoA and finally to succinyl-CoA  Transfer of a methyl group from methyl-tetrahydrofolate (methyl-THF) via Cbl to homocysteine to form methionine — This reaction has two important effects: it reduces the plasma concentration of homocysteine which is probably toxic to endothelial cells; and, perhaps more importantly, it demethylates THF  Demethylation is a critical step in DNA synthesisJune 26, 2012 Total slides : 120 50
    51. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 51
    52. Vitamins & Cofactors Anemia & Vitamins Intense erythroid hyperplasia in the marrow but relative reticulocytopeniaJune 26, 2012 Total slides : 120 52
    53. Vitamins & Cofactors Anemia & Vitamins Symptoms  Loss of appetite  Diarrhea  Numbness and tingling of hands and feet  Paleness  Shortness of breath  Fatigue  Weakness  Sore mouth and tongue  Confusion or change in mental status in severe or advanced casesJune 26, 2012 Total slides : 120 53
    54. Vitamins & Cofactors Anemia & Vitamins Exams and Tests A physical exam may show problems with reflexes or positive Babinski reflex. The following tests will be done:  CBC  Bone marrow examination  LDH  Vitamin B12 level  Schilling test  Antibody test  Methylmalonic acid testJune 26, 2012 Total slides : 120 54
    55. Vitamins & Cofactors Anemia & Vitamins Bone marrow aspiration  A small amount of bone marrow is removed during a bone marrow aspiration. The procedure is uncomfortable, but can be tolerated by both children and adults. The marrow can be studied to determine the cause of anemia, the presence of leukemia or other malignancy, or the presence of some "storage diseases" in which abnormal metabolic products are stored in certain bone marrow cells.June 26, 2012 Total slides : 120 55
    56. Vitamins & Cofactors Anemia & Vitamins Schilling test  The Schilling test is performed to evaluate vitamin B12 absorption. B12 helps in the formation of red blood cells, the maintenance of the central nervous system, and is important for metabolism. Normally, ingested vitamin B12 combines with intrinsic factor, which is produced by cells in the stomach. Intrinsic factor is necessary for vitamin B12 to be absorbed in the small intestine. Certain diseases, such as pernicious anemia, can result when absorption of vitamin B12 is inadequate.June 26, 2012 Total slides : 120 56
    57. Vitamins & Cofactors Anemia & Vitamins LDH Alternative Names  Lactate dehydrogenase; Lactic acid dehydrogenase Definition LDH is a blood test that measures the amount of lactate dehydrogenase (LDH). How the Test is Performed  The health care provider draws blood from a vein or from a heel, finger, toe, or earlobe. The laboratory quickly spins (centrifuges) the blood to separate the serum (liquid portion) from the cells. The LDH test is done on the serum.June 26, 2012 Total slides : 120 57
    58. Vitamins & Cofactors Anemia & Vitamins Antibodies test  Your doctor may draw a sample of your blood to check for antibodies to intrinsic factor. In the majority of cases, vitamin B-12 deficiency is due to a lack of intrinsic factor — a protein secreted by the stomach necessary for the absorption of vitamin B-12. The presence of antibodies to intrinsic factor indicates pernicious anemia.June 26, 2012 Total slides : 120 58
    59. Vitamins & Cofactors Anemia & Vitamins Methylmalonic acid test  You may undergo a blood and urine test to measure the presence of a substance called methylmalonic acid. The level of this substance is higher in people with vitamin B-12 deficiency.June 26, 2012 Total slides : 120 59
    60. Vitamins & Cofactors Anemia & Vitamins Treatment  Treatment depends on the specific cause of B12 deficiency anemia.  Pernicious anemia requires lifelong vitamin B12 injections. Those with anemia due to a lack of vitamin B12 may be told to take vitamin supplements and to follow a more balanced diet. It may be treated initially with vitamin B12 injections.  Anemia caused by malabsorption is treated with vitamin B12 injections until the condition improve  Outlook (Prognosis)  Treatment for this form of anemia is usually effectiveJune 26, 2012 Total slides : 120 60
    61. Vitamins & Cofactors Anemia & Vitamins Prevention Anemia caused by a lack of vitamin B12 can be prevented by following a well-balanced diet. B12 injections can prevent anemia after surgeries known to cause vitamin B12 deficiency. Early diagnosis and prompt treatment can limit the severity and complications of this anemia.June 26, 2012 Total slides : 120 61
    62. Folic Acid & Anemia Anemia & Vitamins
    63. Vitamins & Cofactors Anemia & Vitamins Folate-deficiency anemia  Folate-deficiency anemia is a decrease in red blood cells (anemia) caused by folate deficiency.  The hematologic manifestations of folate deficiency are similar to those of Cbl deficiency but neurologic abnormalities do not occur Symptoms  Tiredness  Headache  Sore mouth and tongue  PallorJune 26, 2012 Total slides : 120 63
    64. Vitamins & Cofactors Anemia & Vitamins Causes  Nutritional deficiency Substance abuse ,Alcoholism Poor dietary intake ,Overcooked foods ,Depressed patients, Nursing homes  Malabsorption Sprue, Inflammatory bowel disease Infiltrative bowel disease,Short bowel syndrome  Drugs (various mechanisms) Methotrexate, Trimethoprim, Ethanol, Phenytoin  Increased requirements Pregnancy, lactation, Chronic hemolysis, Exfoliative dermatitisJune 26, 2012 Total slides : 120 64
    65. Vitamins & Cofactors Anemia & Vitamins Folate metabolism DNA dTMP DHF PG THF PG 5,10 dUMP METHYLENE THF PG THF HOMOCYSTEINE METHIONINE SYNTHASE METHIONINE METHYL THFJune 26, 2012 Total slides : 120 65
    66. Vitamins & Cofactors Anemia & Vitamins Exams and Tests  Low red blood cell folate level.  A complete blood count (CBC) shows anemia and large red blood cells.  A bone marrow examination is rarely necessary, but shows megaloblasts.June 26, 2012 Total slides : 120 66
    67. Vitamins & Cofactors Anemia & Vitamins Treatment  The goal is to treat the cause of the anemia, which may be poor diet or a malabsorption disease.  Oral or intravenous folic acid supplements may be taken on a short-term basis until the anemia has been corrected, or -- in the case of poor absorption by the intestine -- replacement therapy may be lifelong.  Dietary treatment consists of increasing the intake of green, leafy vegetables and citrus fruits. Outlook (Prognosis)  Anemia usually responds well to treatment within 2 months.June 26, 2012 Total slides : 120 67
    68. Vitamins & Cofactors Anemia & Vitamins Possible Complications  Symptoms of anemia can cause discomfort. In a pregnant woman, folate deficiency has been associated with neural tube or spinal defects (such as spina bifida) in the infant.June 26, 2012 Total slides : 120 68
    69. Vit amin A & Anemia Anemia & Vitamins
    70. Vitamins & Cofactors Anemia & Vitamins Vitamin A & anemia  In many developing countries vitamin A deficiency (VAD) is considered to be a major public health problem and concurrently the prevalence of anemia is high in populations affected by VAD.  190-255 millions preschool-aged children throughout the world are vitamin A deficient, with some 3–5 million having xerophthalmia, and 500 000 becoming blind and dying each year. Vitamin A deficiency may be responsible for 25–35% of all early childhood deaths in high risk regions of the developing world, attributed to increased severity of infection in a deficient state.June 26, 2012 Total slides : 120 70
    71. Vitamins & Cofactors Anemia & Vitamins Animal studies Vitamin A-deficient rats indicate:  Losses of hematopoietic tissue in bone marrow.  Splenic accumulation of hemosiderin. Adding of vitamin A to the rats diet:  Regeneration of the bone marrow.  Disappearance of hemosiderin from the spleen.  Enhanced erythroblastic activity.June 26, 2012 Total slides : 120 71
    72. Vitamins & Cofactors Anemia & Vitamins Human studies In human studies:  Positive correlation between serum retinol concentration and Hb level.  The findings suggest that adequate vitamin A status can help maintain adequacy of plasma iron to supply body tissues.  Intake of fortified food items with vitamin A has been resulted in elevation serum iron levels,transferrin saturation and serum ferritin levels.  Result: increasing iron availability to tissues.June 26, 2012 Total slides : 120 72
    73. Vitamins & Cofactors Anemia & Vitamins  Vitamin A appears to be involved in the pathogenesis of anemia through diverse biological mechanisms, such as:  The enhancement of growth and differentiation of erythrocyte progenitor cells  Potentiation of immunity to infection  Reduction of the anemia of infection  Mmobilization of iron stores from tissues.  Epidemiological surveys show that the prevalence of anemia is high in populations affected by vitamin A deficiency in developing countries. Improvement of vitamin A status has generally been shown to reduce anemia.June 26, 2012 Total slides : 120 73
    74. Vitamins & Cofactors Anemia & Vitamins A combination of vitamin A with iron and zinc is more effective than with iron alone.  This could reflect Zn association with increases in plasma vitamin A and retinol-binding protein.  The effect of vitamin A on risk of anemia appears to be more variable in pregnancy than childhood.  Haemoconcentration associated with low vitamin A status can mask anemia.June 26, 2012 Total slides : 120 74
    75. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 75
    76. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 76
    77. Vitamins & Cofactors Anemia & Vitamins  It has been reported that supplementation with vitamin A increases hemoglobin levels and packed cell volumes in humans with low vitamin A status, thereby contributing to the control of nutritional anemia; furthermore, a synergistic interaction exists between vitamin A and iron in combined therapy (Suharno et al., 1993). Some important effects of vitamin A are to support erythropoiesis in the bone marrow and to mobilise iron from body stores (Bloem, 1995; Roodenburg et al., 1996; Semba and Bloem, 2002). However, in rats and chickens vitamin A deficiency (VAD) is accompained by imbalances in water regulation, in particular a decrease in extracellular water, which may lead to hemoconcentration as the VAD proceeds (Sure et al., 1929; McLaren et al., 1965; Nockels and Kienholz, 1967; Corey and Hayes, 1972; Mejı´a et al., 1979a; Roodenburg et al., 1994, 1996).June 26, 2012 Total slides : 120 77
    78. Vitamins & Cofactors Anemia & Vitamins Possibly mechanism of vitamin A deficiency anemia Vitamin A deficiency may induce anemia by:  Impairing the differentiation and proliferations of pluripotent haematopoietic cells.  Disturbing renal and hepatic erythropoietin synthesis.  Disturbing GI absorption.  Reducing mobilization of body iron stores..June 26, 2012 Total slides : 120 78
    79. Vit amin K & Anemia Anemia & Vitamins
    80. Vitamins & Cofactors Anemia & Vitamins Vitamin K and hemolytic anemia  Vitamin K is necessary for synthesis in the liver of factor II (prothrombin), factor VII (proconvertin), factor IX (thromboplastin), and factor X. Deficiency of vitamin K or disturbances of liver function may lead to deficiencies of these factors. When the prothrombin level falls to about 10 to 15% of normal, even slight trauma may cause bleeding; when the level is below 10%, spontaneous hemorrhage may occur, in the form of hematoma, hematemesis, hematuria or melena. The mechanism by which vitamin K promotes formation in the liver of clotting factors II, VII, IX, and X is not known.June 26, 2012 Total slides : 120 80
    81. Vitamins & Cofactors Anemia & Vitamins  Newborns should be observed for vitamin K deficiency. The incidence of vitamin K deficiency is higher in breast-fed infants.  In newborns, particularly premature infants, hyperbilirubinemia and hemolytic anemia have been reported. The risk is much less with phytonadione than other vitamin K preparations unless high doses (10 to 20 mg) are given.  In infants (particularly premature babies), excessive doses of vitamin K analogs during the first few days of life may cause hyperbilirubinemia; this in turn may result in severe hemolytic anemia, hemoglobinuria, kernicterus, leading to brain damage or even death.June 26, 2012 Total slides : 120 81
    82. Vitamins & Cofactors Anemia & Vitamins Hemorrhagic Disease of the Newborn: Prophylaxis:  In its 1997 clinical practice guidelines on vitamin K administration, the Canadian Paediatric Society recommends that vitamin K1 be given as a single i.m. injection to all newborns within 6 hours of birth, at a dose of 1 mg for infants with a birthweight of >1 500 g and 0.5 mg if birthweight is £1 500 g.June 26, 2012 Total slides : 120 82
    83. Vitamins & Cofactors Anemia & Vitamins Vitamin K deficiency  Vitamin K deficiency may occur in patients with biliary obstruction or other conditions limiting absorption of vitamin K such as celiac disease, ulcerative colitis, sprue, regional enteritis, cystic fibrosis, intestinal resection, and in patients receiving drugs that may affect liver function or intestinal flora.June 26, 2012 Total slides : 120 83
    84. Vit amin E & Anemia Anemia & Vitamins
    85. Vitamins & Cofactors Anemia & Vitamins Vitamin E & hemolytic anemia  Hemolytic anemia results from the deficiency of the enzyme glucose-6-phosphate dehydrogenase or of glutathione synthetase. Red blood cells become more sensitive to attack by free radicals, because they cannot form lipids in which vitamins can be stored. Increasing the blood level of vitamin E has been found to be useful in this disease. Function : as an antioxidant, scavenging highly reactive free radicals and protecting the PUFAs of cellular membranes from oxidative destruction.June 26, 2012 Total slides : 120 85
    86. Vitamins & Cofactors Anemia & Vitamins Vitamin E and membrane lipid oxidation As part of the antioxidant network, a-tocopherol (a-OH) forms a tocopheroxyl radical (a-TO·) when it intercepts a peroxyl radical (ROO·) in a cell membrane. In the absence of vitamin E, these ROO· can abstract a hydrogen from PUFA (RH) and generate both a hydroperoxide (ROOH) and another carbon-centered radical (R·), which in the presence of oxygen (O2) will form a ROO· and thus a lipid peroxidation chain reac-tion occurs. If a-tocopherol (a-TOH) is present it intercepts the radical 1000 times faster than the radical reacts with PUFA, and both a ROOH and an a- TO· are formed. This a-TO· radical can be detoxified and a-TOH regenerated by intracellular antioxidants including vitamin C, glutathione, and reducing equivalents (NAD(P)H) derived from oxidative metabolism.June 26, 2012 Total slides : 120 86
    87. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 87
    88. Vitamins & Cofactors Anemia & Vitamins Animal studies Animal studies have observed:  The development of severe anemia in primates.  Morphological abnormalities of the bone marrow among primates. Treatment with vitamin E improved blood parameters among these animals.June 26, 2012 Total slides : 120 88
    89. Vitamins & Cofactors Anemia & Vitamins Human studies  Pre term and LBW infants are born with low serum and tissue concentration of vit E.  Vit E deficiency induced anemia in infants has been characterized by red blood hemolysis and oedema that resolves promptly following vit E treatment.  Vit E is routinly given to preterm infants in developed country to protect against the potential oxidative caused by iron supplementation  Increasing tocopherol to PUFA ratio to lower oxidant agents such as iron in infant formula.June 26, 2012 Total slides : 120 89
    90. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 90
    91. Vitamins & Cofactors Anemia & Vitamins Vitamin E & fanconi anemia  a-Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%).June 26, 2012 Total slides : 120 91
    92. Vitamins & Cofactors Anemia & Vitamins Effects of vitamin E on heme synthesis  Vitamin E has a stimulatory effect on heme synthesis, apparently through its action on ALAD and on 5- aminolevulinic acid synthetase (ALAS).  Addition of vitamin E to the diets of lead-intoxicated rabbits coused a diminution in the anemia and coproporphyrinuria, which had resulted from plumbism.(Nair et al. , deRosa)June 26, 2012 Total slides : 120 92
    93. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 93
    94. Vitamins & Cofactors Anemia & Vitamins Vitamin E deficiency  Vitamin E deficiency can result from a low intake of fresh fruit and vegetables and other foods rich in vitamin E Deficiency can also occur in those individuals who cannot absorb fat. In addition, damage to the pancreas, bile duct, liver, and surgical removal of the major portion of the digestive tract can cause vitamin E deficiency. The plasma level of vitamin E in normal adults is about 10 mcg/ml; a plasma level of 5 mcg/ml or less is considered and indication of vitamin E deficiency.June 26, 2012 Total slides : 120 94
    95. Vit amin C & Anemia Anemia & Vitamins
    96. Vitamins & Cofactors Anemia & Vitamins Vitamin C effects on anaemia  Vitamin C defciency has been associated with various forms of anemia, but it is still unclear whether vitamin C (ascorbate) is directly involved in hematopoiesis or if anemia arises indirectly through the interactions of vitamin C with folate and iron metabolism. In its role as a reducing agent, vitamin C can facilitate iron absorption from the gastrointestinal tract and enable its mobilization from storage.  Iron and ascorbate form an iron chelate complex that is more soluble in the alkaline environment of the small intestine and, as a result, more easily taken up.June 26, 2012 Total slides : 120 96
    97. Vitamins & Cofactors Anemia & Vitamins  Vitamin C may counteract the inhibition of iron absorption by dietary phytates and tannins.  Ascorbic acid also activates the enzyme folic acid reductase to form THF ,the active form of folate which prevent megaloblastic anemia.  Vitamin C possibly prevent hemolysis resulting from compromised celluar antioxidant defence mechanism.June 26, 2012 Total slides : 120 97
    98. Vitamins & Cofactors Anemia & Vitamins Vitamin C mechanism Ultimately Vitamin C may:  improve absorption of non-heme iron  protect against oxidative damage  counteract the effects of iron absorption inhibitors.  increase serum iron , ferritin and Hb concentrations among children and non-pregnant subjects.June 26, 2012 Total slides : 120 98
    99. Vitamins & Cofactors Anemia & Vitamins Vitamin C deficiency  Vitamin C deficiency is evident when serum ascorbate falls below 11.4 mmol /1. Groups that have been identifed as being at risk of vitamin C deficiency include pregnant and lactating women, infants fed exclusively cows milk, elderly men and smokers.June 26, 2012 Total slides : 120 99
    100. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 100
    101. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 101
    102. Biotin & Anemia Anemia & Vitamins
    103. Vitamins & Cofactors Anemia & Vitamins  Biotin is one of the least-studied vitamins, particularly in relation to mitochondrial function and the extent of its nutritional deficiency in humans.  The most important function of Biotin is to ensure proper growth. Not only does it help produce DNA fatty acids and other essential nucleic acids, it also helps the cells grow and replicate. It also plays a vital role in the production of bone marrow and thus the tissues of the central nervous system and muscles benefit from this vitamin. Vitamin H is also known to be involved in the process that helps transfer carbon dioxide.June 26, 2012 Total slides : 120 103
    104. Vitamins & Cofactors Anemia & Vitamins Effects of biotin on heme synthesis  Biotin is a coenzyme in 5 different biotin-dependent carboxylases (BDC), which catalyze carboxylation reactions : pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), acetyl- CoA carboxylase (ACC)-2, and ACC-1. The first 4 are located in the mitochondria. PC, PCC, and MCC catalyze anaplerotic reactions and replenish tricarboxylic acid (TCA) cycle intermediates .June 26, 2012 Total slides : 120 104
    105. Vitamins & Cofactors Anemia & Vitamins Effects of biotin on heme synthesis  BD has a detrimental effect on the level of TCA cycle intermediates. A deficiency in PC directly decreases production of oxaloacetate. A deficiency in PCC decreases production of succinyl-CoA and causes propionyl-CoA to accumulate, which interacts via a side reaction with oxaloacetate to form methylcitrate. Additionally, low activity of MCC causes methylcrotonyl-CoA to accumulate in the mitochondria where it reacts with glycine and potentially depletes this amino acid from the mitochondrial matrix.  Succinyl-CoA from the TCA cycle and glycine are the precursors for heme biosynthesis. Heme synthesis starts in the mitochondria by condensing succinyl-CoA with glycine to form -aminolevulinate, the first metabolite committed to heme synthesisJune 26, 2012 Total slides : 120 105
    106. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 106
    107. Vitamins & Cofactors Anemia & Vitamins Results  Heme level and synthesis were markedly decreased in BD cells , indicating that adequate heme synthesis requires biotin and that BD can cause heme deficiency. Thus, biotin should be considered the 8th member of the group of vitamins and minerals required for adequate heme synthesis . The decrease in iron uptake in BD cells is unexpected, because heme deficiency should be expected to cause a compensatory increase in iron uptake . A possible explanation for the lack of an increase in iron uptake in BD cells is that the heme deficiency caused by BD is due to a decrease in succinyl- CoA, which lowers the production of porphyrins. Porphyrins are intermediates in the biosynthesis of heme. These results suggest that optimal uptake of iron requires that the mechanisms for iron assimilation into heme remain intact. Adequate levels of biotin appear to be essential for adequate iron uptake. Thus, for correcting iron deficiency in humans, it may be important to ensure biotin adequacy.June 26, 2012 Total slides : 120 107
    108. Orotic acid & Anemia Anemia & Vitamins
    109. Vitamins & Cofactors Anemia & Vitamins  Orotic acid plays a central role in the metabolism of folic acid and vitamin B-12, and may enhance the transportation of minerals across cell membranes.  Orotic acid and folate are also involved in DNA synthesis. Many of the vitamin-like effects of orotic acid are undoubtedly due to its role in RNA and DNA synthesis. Our bodies produce OA as an intermediate in the manufacture of the pyrimidine bases uracil, cytosine, and thymine. Together, these pyrimidines constitute half of the bases needed for RNA/ DNA, the other half coming from the purine bases adenine and guanine which are synthesized independently of orotic acid.June 26, 2012 Total slides : 120 109
    110. Vitamins & Cofactors Anemia & VitaminsJune 26, 2012 Total slides : 120 110
    111. Vitamins & Cofactors Anemia & Vitamins Mechanism of OA action  The oral administration of the pyrimidine precursor orotic acid in doses of 3 to 6 Gm. daily to patients with pernicious anemia in relapse produced with some regularity Partial remissions in the manifestations of vitamin B12 deficiency.  The early effects of orotic acid in pernicious anemia resembled those of small amounts of B12. Reticulocytosis appeared 7 to 14 days after the start of therapy.June 26, 2012 Total slides : 120 111
    112. Vitamins & Cofactors Anemia & Vitamins Mechanism of OA action  B12 is concerned with pyrimidine biosynthesis.  The degree of remission that can be produced in patients with pernicious anemia in relapse by the administration of orotic acid suggests, that one major consequence of vitamin B12 deficiency in the human is a defect in pyrimidine biosynthesis and/or incorporation. Other processes, such as purine ring formation, may also be affected. The mechanism by which orotic acid induces partial remissions in pernicious anemia is unknown.June 26, 2012 Total slides : 120 112
    113. Vitamins & Cofactors Anemia & Vitamins Mechanism of OA action  It could serve merely as a metabolite which when supplied from exogenous sources would circumvent a block in its synthesis or in that of a precursor. Increasing the supply of orotic acid could possibly overcome by mass action a defect in the synthetic pathway at a later stage. In view of demonstrated feed-back regulatory mechanisms in pyrimidine synthesis,June 26, 2012 Total slides : 120 113
    114. Other vit amins & Anemia Anemia & Vitamins
    115. Vitamins & Cofactors Anemia & Vitamins PABA Para-aminobenzoic acid, as part of the coenzyme tetrahydrofolic acid, aids in the metabolism and utilization of amino acids and is also supportive of blood cells, particularly the red blood cells. PABA supports folic acid production by the intestinal bacteria.June 26, 2012 Total slides : 120 115
    116. Vitamins & Cofactors Anemia & Vitamins Inositol  Usually considered part of the vitamin B complex. It is thought that along with choline, inositol is necessary for the formation of lecithin within the body. Involved in calcium mobilization.  IP6 regulates the oxygen capacity of red blood cells; it reduces both cholesterol and trigylcerides, as well as preventing heart damage during a heart attack.  Research has shown that IP6 can help prevent sikle cell anemia  Anemia has been reported as a clinical sign of inositol deficiency in salmonids (Halver, 1982). Waagbø et al. (1998) observed a positive correlation between blood hemoglobin concentrations and dietary levels of inositol in Atlantic salmon.June 26, 2012 Total slides : 120 116
    117. Vitamins & Cofactors Anemia & Vitamins adenine  Acts as a co-enzyme with other vitamins to enhance metabolism.  Acts as a precursor for assimilation of other B-vitamins.  Strengthens the immune system response.  Promotes cell formation and normal growth.  Prevents cellular mutation and free radical formation.  Helps to balance blood sugar levels.  Deficiency of adenine associated whit blood and skin disordersJune 26, 2012 Total slides : 120 117
    118. Vitamins & Cofactors Anemia & Vitamins Vitamin DJune 26, 2012 Total slides : 120 118
    119. Vitamins & Cofactors Anemia & Vitamins  The association of hypercalcemia and anemia suggested a neoplastic origin; this idea was rejected when results of additional examinations became available. High vitamin D levels could directly affect hematopoietic cells or act through high calcium levels, which inhibit erythroid colony formation in vitro and erythropoietin production in vitro and in vivo. That calcium is more important than vitamin D itself is supported by the course of our patient, whose anemia subsided after normalization of calcium levels, despite high vitamin D levels.  In addition to the danger of extemporaneous formulations, which carry a higher risk for error than factory-made pills, anemia is another potential complication of vitamin D intoxication.June 26, 2012 Total slides : 120 119
    120. Vitamins & Cofactors Anemia & Vitamins Laetrile  Relationship between laetrile and anemia…  What is your idea?June 26, 2012 Total slides : 120 120
    121. Thank you Questions ?

    ×