Trace Elements CC lab PDF

# Trace Elements **organic** -> vitamins **inorganic** -> trace metals - Also known as **MICRONUTRIENTS ** - naturally occurring, homogeneous - inorganic substance required in humans in amounts less than 100 mg/day - have specific *in vivo* metabolic functions which cannot be effectiv...

# Trace Elements **organic** -> vitamins **inorganic** -> trace metals - Also known as **MICRONUTRIENTS ** - naturally occurring, homogeneous - inorganic substance required in humans in amounts less than 100 mg/day - have specific *in vivo* metabolic functions which cannot be effectively performed by other elements ## Categories Elements are categorized based upon their biological effect, diseases that occur due to their deficiency and toxicity due to overdose. 1. **Essential elements** - deficiency impairs a biochemical reaction and replacement of the element corrects the impairment 2. Possibly essential elements 3. **Nonessential elements** - primarily of medical interest because of their **TOXICITY**. - Usually consists of **metals** except **selenium** and the halogens **fluoride** and **iodine** ## Roles of Trace Elements 1. Structural signal transduction and special catalytic properties - Zn, Cu, Mg 2. Some trace elements are components of metalloenzymes and metalloproteins acting as cofactors - Fe, Se, Mo, Cr 3. Provides electron and oxygen transport - Fe, Cu, Se 4. Maintenance of macromolecule conformation - Zn, Cu 5. Influences vitamin or hormonal activity - I, Zn, Cr, Se ## Possibly essential in some animals - Bromine - Cadmium - Lead - Strontium - Tin ## Not essential - Aluminum - Antimony - Bismuth - Germanium - Mercury - Silver - Thallium - Titanium ## 1. Aluminum - non-essential - Crystalline silver-white ductile metal - **MOST ABUNDANT METAL IN THE EARTH’S CRUST** - Good conductor of heat and electricity - Found in consumer products such as antacids, astringents, buffered aspirin, food additives, cosmetics, and antiperspirants ### Aluminum - Mode of exposure: orally, through inhalation and parenterally - inject - In plasma, aluminum is bound to carrier proteins such as transferrin due to chemical similarity with iron interms of ionic charge (+3) - Concentration in organs: - Bone: 50% - Lung tissues: 25% - Excretion - Urine: 95% - Bile: 2% ### Lab Evaluation - **Specimens:** - Urine - Serum - **Methodologies:** - ICP-MS - GFAAS ### ICP-MS : Inductively coupled plasma mass spectrometry - This is a diagram of an Inductively coupled plasma mass spectrometry (ICP-MS). It shows a gas cylinder, a gas inlet, a plasma torch, an interface, a quadrupole, an ion lens, a detector, and a nebulizer. The nebulizer injects a sample into the plasma. The plasma is a very high temperature gas that ionizes the elements in the sample. The ions are then passed through a quadrupole, which separates them by their mass-to-charge ratio. The ions that are detected at the detector are used to determine the concentration of each element in the sample. ### GFAAS: Graphite furnace atomic absorption spectroscopy - This is a diagram of a Graphite furnace atomic absorption spectroscopy (GFAAS). It shows a light source, an atomization chamber, a monochromator, and a detector. The sample is placed in the atomization chamber. The atomization chamber heats the sample, causing the elements in the sample to become atoms. The atoms absorb light from the light source. The amount of light absorbed is proportional to the concentration of the element in the sample; at 3000 degrees Celsius, the elements will become atoms. ## 2. ARSENIC - NONESSENTIAL - **Food** is the largest source of arsenic exposure, with lower amounts coming from drinking water and air - Most commonly found in **fish** and **seafood** - Mainly used as a wood preservative ## 10. IRON - Ferrous (Fe2+) and ferric (Fe3+) irons - Fourth most abundant element in the earth's crust and the most abundant transition metal - **No excretory system is used for excess iron** - Primary means of iron regulation - Absorption of iron from the intestine ### Iron Metabolism - Only the **Fe2+** form can be absorbed by the intestine - Fe3+ is first reduced to Fe2+ by **vitamin C** or **ferric reductases** present in the intestinal epithelium. - In the intestinal cells, Fe2+ may be stored as ferritin or oxidized to Fe3+ or exported in the basolateral side, and bound to **apotransferrin** for distribution in the body - Release of Fe3+ from intestinal cells is mediated by **ferroportin** and regulated by **hepcidin** ### Iron - Of the 3-5 g of iron in the body, approximately 2-2.5 g of iron is in **hemoglobin**, mostly in RBC and its precursors. - A moderate amount of iron (130mg) is found in **myoglobin** -> skeletal muscle - A small 8mg is found in enzymes like peroxidases, cytochromes and many of Kreb cycle's enzymes - Iron is stored as **FERRITIN OR HEMOSIDERIN** primarily in the bone marrow, spleen or liver. - Hemosiderin is formed when ferritin is broken down. - Only 3-5 mg of iron is found in plasma bound to transferrin, albumin and free hemoglobin ### Iron Deficiency - Affects 15% of the worldwide population - At risk: - Pregnant women - Young children - Adolescents - Women of reproductive age - Caused by increased blood loss, decreased intake or decreased release from ferritin - **IDA - MOST COMMON ANEMIA IN THE PLANET** - **Other features of IDA:** - Pagophagia - craving ice - Pica - craving of nonfood substances (e.g., dirt, clay, laundry starch) - Glossitis - smooth tongue - Restless legs - Angular stomatitis - cracking of corners of mouth - Koilonychia - thin, brittle, spoon-shaped fingernails ### Iron Toxicity - **Hemochromatosis** - collective term for iron overload **with or without** tissue damage. - associated hereditary hemochromatosis (HH) leading to abnormally high iron absorption - affects liver function, and often leads to hyperpigmentation of the skin - **Hemosiderosis** - iron overload **without** a demonstrable tissue damage. This is a diagram of healthy liver vs Hemochromatosis liver. The healthy liver is brown with a white line in the middle. The Hemochromatosis liver is more orange with a white line in the middle and a red streak pointing to a micro-lesion in the red center of the image. ### Lab evaluation - Disorders of iron metabolism are evaluated by: - Hematocrit & hemoglobin - RBC count & RBC indices - Total iron level - Percent saturation - Transferrin - Ferritin - TIBC (Total iron-binding capacity) ### Total Iron Content (Serum Iron) - Refers specifically to the **Fe3+ bound to transferrin** - Heparinized plasma may be used - Oxalate, citrate, EDTA binds iron and are all unacceptable - **Early morning sample** is preferred because of diurnal variation ### Total Iron Binding Capacity (TIBC) - Refers to the amount of iron that could be bound by **saturating transferrin** and other minor-iron binding proteins present in the serum or plasma sample. - Direct measure of the available transferrin receptors - All TIBC methods require addition of excess iron to saturate transferrin - Excess iron is removed by adding **magnesium carbonate** to measure the bound iron. - IDA have **high TIBC**, non-iron deficiency anemias have **low TIBC.** ### Percent Saturation - It is also known as the **transferrin saturation** - **AN INDEX OF IRON STORAGE** - It is the ratio of serum iron to TIBC - This ratio is around 1/3 for normal individuals, and in IDA it is significantly reduced to values of around 1/5 or lower - Increased: iron overdosage, hemochromatosis, sideroblastic anemias - Decreased: IDA (lowest), malignancy, chronic infection, anemia of chronic disease. - **Reference values** - 20-55% (conventional) - %saturation = total iron (ug/dL)/TIBC x 100 ### Lab Methodologies 1. **COLORIMETRY** - Uses HCI and ferrozine —> (+) blue color - Iron dyes: bathophenanthroline, tripyridyltriazine 2. **ANODIC STRIPPING VOLTAMMETRY** - **Notes:** - The first step in quantitation of serum iron is separation from transferrin. - Serum iron is falsely elevated by hemolysis and affected by diurnal variation. - IRON IS HIGHEST IN THE MORNING AND LOWEST AT NIGHT ## 11. Lead - Lead plays **no known role** in normal human physiology - Lead-based household paints was banned in the US in 1972. - In recent years, there has been massive recall of toys from China. - Exposure is primarily gastrointestinal and respiratory - 99% of absorbed lead is taken up by erythrocytes where it **interferes with heme synthesis** ### Lead Poisoning - Lead blocks the action of: - 6-aminolevulinic acid (ALA) synthetase - 6-ALA dehydratase (ALAD) - coproporphyrinogen decarboxylase - Ferrochelatase - **-> producing ANEMIA** - In addition, lead blocks two other enzymes: - pyrimidine-5'-nucleotidase - Na-K dependent ATPase - resulting in diminished energy supply for red blood cells, leading to decreased cell membrane integrity. - Because **pyrimidine-5'-nucleotidase** is required for removal of clumped intracellular RNA, lead inhibition of this enzyme results in clumping of RNA complexes, giving rise to the observed **BASOPHILIC STIPPLING OF RED BLOOD CELLS** This is a micrograph of **Coarse Basophilic Stippling**, it shows a red circle around a red blood cell with many purple dots. This is a finding that is indicative of **lead poisoning** ### Lab Methodologies - **Specimen: WHOLE VENOUS BLOOD** - Preferred over serum or plasma since majority of lead is found inside red cells - **Urine:** useful for detecting RECENT exposure or monitoring of chelation therapy - **Radiographic methods:** measure amount of lead in bones - **Others:** plasma aminolevulinic acid, whole blood Zinc protoporphyrin, free erythrocyte protoporphyrin ## 12. Mercury - Also called **quicksilver** - Three naturally occurring oxidation states: - Hg(0), Hg+, Hg2+ - **Organic mercury:** Hg2+ attached to carbon atoms - Mercury is used in dental amalgams, electronic switches, germicides, fungicides and fluorescent light bulbs - OTC drugs like, topical antiseptics, stimulant laxatives, diaper-rash ointment, eye drops, nasal sprays and eye cosmetics, especially **mascara** ### Mercury - **Routes of exposure:** - Inhalation, primarily elemental mercury vapor - Ingestion of HgCl2 and mercury-containing foods such as predatory fishes - Cutaneous absorption of methyl mercury through the skin and even through latex gloves - Injection of liquid mercury and mercury-containing tattoo pigments - Dental amalgams ### Forms of Mercury - Elemental mercury vapors: highly absorbed and highly toxic; harmful effects on nervous, digestive and immune systems. - Liquid elemental mercury: poorly absorbed and relatively nontoxic - Inorganic salts: affects the skin, eyes, GIT, and kidneys - Methyl mercury: very toxic, highly selective for lipid-rich medium such as brain ## 13. Molybdenum - An essential trace element which is a component of xanthine oxidase, aldehyde oxidase and sulfite oxidase - Binds molybdenum in the form of a cofactor called **molybdopterin** - Grains, nuts, and legumes such as peas, lentils, and beans are good sources - Can cross the placental barrier and increase intake of molybdenum in the diet of the mother, which can increase its level in the neonate's liver ## 14. Selenium - Considered as a toxic element in the 1930s, carcinogen in 1940s, essential element in 1950s and anticarcinogen since 1960s - A constituent of **glutathione peroxidase** that is associated with **vitamin E** in its functions, which is important in defense against oxidative stress - **antioxidant**. - Also involved in the metabolism of thyroid hormones deiodinase, thioredoxin reductase ### Selenium - **Deficiency** is associated with **cardiomyopathy**, skeletal muscle weakness and **osteoarthritis** - **Keshan disease** - an **endemic cardiomyopathy** that affects mostly children and child-bearing age in certain areas in China is associated with selenium deficiency. - **Kashin–Beck disease** - **endemic osteoarthritis** that occurs during adolescent and preadolescent years, another disease linked to low selenium levels in Norther China, North Korea and Eastern Siberia. - **Toxicity:** hair loss, garlic breath, irritability, mild nerve damage, and nail damage ## 15. Zinc - **Second** only to iron in importance as an essential trace element - The **most common catalytic metal ion** in the cytoplasm of cells - **Main biochemical role of zinc** is seen in its influence on the **activity of more than 300 enzymes** in classes such as oxidoreductases, transferases, lyases, isomerases and lipases. ### Genetic Zinc Deficiency - **ACRODERMATITIS ENTEROPATHICA** - A rare autosomal recessive disorder with impaired intestinal absorption and transport of zinc. - **Symptoms include:** - **hyperpigmented skin lesions**, pustular and bullous dermatitis, alopecia, growth retardation, diarrhea, secondary infection, irritability, lethargy, and depression ### Zinc Toxicity - Inhalation of zinc oxide fumes is the most common cause of METAL FUME FEVER - Chronic oral zinc supplementation **interferes with copper absorption** and may cause copper deficiency, forming the basis for using zinc to treat Wilson's disease. ## Analytical Methodologies ## Analytical Considerations - Assessment of trace mineral status is difficult and requires **specialized analytical instruments** (e.g, AAS, AES) - Serum measurements are complicated by **associated disease states** that affect levels of circulating binding proteins (e.g., albumin) - Diagnosis is dependent on **high degree of suspicion**, **careful inspection for signs and symptoms**, **thorough understanding of predisposing causes** and **resolution of symptoms** with therapeutic trial - **Assessment of trace element status** requires measurement of either the **CONCENTRATION** in accessible tissues (hair, nails) and body fluids (serum, urine) or the ACTIVITY OF A TRACE ELEMENT-dependent enzyme. - Hair and nail clippings should be collected with care and washed to avoid surface contamination. - Assays in blood, serum, or urine usually reflect the current nutriture or RECENT exposure - Hair, fingernail, or toenail analyses provide an assessment of CHRONIC exposure - There are no particularly good indicators for the determination of trace element dietary status because of poor correlation with total body stores. - The only **definitive test** of human trace mineral element deficiency is: **CLINICAL RESPONSE TO CONTROLLED SUPPLEMENTATION** ## Specimen Collection & Processing - Trace elements must be analyzed with considerable care - Clean room techniques and **ultra-pure reagents** must be used. - Type I water - Reference materials and strict quality control are requisite with each assay run to ensure analytic accuracy. - Special collection and handling are necessary. - They are ubiquitous. - Materials of biological devices (e.g., needles, syringes, stoppers) can readily contaminate a sample. - **Remedy:** use trace element-free syringes - Evacuated tubes with fitted siliconized needles, acid-washed glassware - Use standard reference materials with certified values ## Causes of Deficiency - **Decreased intake** -> nutritional deficiency - **Total parenteral nutrition** -> inadequate supplementation - **Increased utilization** -> increased catabolism - **Interactions between trace elements** (zinc, copper) or with other nutrients (zinc, vitamin C). - Zinc interferes with copper absorption resulting in copper deficiency and anemia. - **Increased excretion** - **Disease states** < insufficient intestinal absorption - Acquired: malabsorption syndromes, intestinal resection - Genetic: mutation resulting in the lack of protein needed to absorb the element - Includes: - Menkes' kinky hair syndrome (copper) - Congenital atransferrinemia (iron) - Acrodermatitis enteropathica (zinc) - Xanthine and sulfite oxidase deficiencies (molybdenum) ### Lab Methodologies 1. **ATOMIC ABSORPTION SPECTROMETRY** - most commonly used for trace and toxic metal analysis - **Flame AAS:** Uses flame; used in the measurement of copper, iron, zinc. - LOQ (limit of quantitation) parts per million (mg/L) - **GFAAS:** Uses graphite furnace, used in the measurement of selenium, cadmium, lead. ### Atomic Absorption Spectroscopy - The **most widely used instrument** for clinical trace element analysis in biological samples - Analytic procedure for the quantitative determination of elements through the **absorption of optical radiation by FREE ATOMS on the gas phase** - **Four important components** 1. Radiation (light) source - emits the spectrum of the analyte element 2. Atomizer - in which the atoms of the element of interest in the sample are formed. 3. Monochromator - for the spectral dispersion of the radiation and separation of the analytical line from other radiation 4. Detector ### Lab Methodologies 2. **INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS)** - Highly sensitive and specific method for measurement of multiple trace elements in a single run - Uses ionized gas (argon) to excite the atoms - Uses internal standard Yttrium 3. **ATOMIC EMISSION SPECTROSCOPY (AES)** - Three components: - Source: Flame or inductively coupled plasma. Liquid sample is converted into aerosol and delivered into the source where sufficient energy will excite the atoms. - Monochromator: Isolates specific wavelengths - Detector: Measures the intensity of radiation. Photographic film (obsolete), replaced by photomultipliers and array-based detector systems ### Elemental Speciation - Toxicity of the elements may depend on their chemical form - e.g. Arsenic, Arsenobetaine: nontoxic form of arsenic, Methylated forms: intermediate in toxicity, Inorganic arsenic (As[V], As[III]): highly toxic - Hyphenated techniques - Allows determination of elemental species - Combination of two or more complementary analytical techniques This is a diagram of an ICP-MS system. The system consists of an Radio-Frequency (RF) generator, a gas cylinder, an autosampler, a nebulizer, a spray chamber, a plasma torch, a mass spectrometer, and a computer. The sample is introduced into the system via the autosampler. The nebulizer creates an aerosol of the sample. The aerosol is then passed through the plasma torch, which ionizes the atoms. The ionized atoms are then passed through the mass spectrometer, which separates them according to their mass-to-charge ratio.