Trace Elements CC Lab Lecture Notes PDF

# Trace elements - 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 can not be effectively performed by other elements. ## Categories - Elements...

# Trace elements - 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 can not 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 | Not Essential | |:---------------------------------------:|:--------------------:| | Bromine | Aluminum | | Cadmium | Antimony | | Lead | Bismuth | | Strontium | Germanium | | Tin | Mercury | | | Silver | | | Thallium | | | Titanium | ## 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 in terms 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 - **Diagram**: There is a diagram of the instrument showing a plasma torch, an interface, a quadrupole, an ion lens, and a detector. The diagram also shows the flow of carrier gas, auxiliary gas, and plasma gas. There is a handwritten note indicating that "elements will became ions" by the interface at 10,000 K. There is also a handwritten note indicating that "mass to charge ratio" is measured by the quadrupole. ### GFAAS: Graphite Furnace Atomic Absorption Spectroscopy - **Diagram**: There is a diagram of the instrument showing a light source, an atomization chamber, a monochromator, and a detector. There is also a handwritten note indicating that "elements will become atom" by the atomization chamber. There is also a handwritten note indicating that the atomization chamber is heated at 3,000 °C. ## 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.** ## Iron - **Ferrous (Fe2+) and ferric (Fe3+) ions** - 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+ 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 (130 mg) is found in **myoglobin** (skeletal muscle). - A small **8 mg** 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 (Iron Deficiency Anemia) - 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. - **Diagram**: There are two diagrams of livers. The first diagram is labeled a "healthy liver" and the second diagram is labeled "hemochromatosis liver". Both livers are shown with arrows pointing to the tissue. The tissue of the hemochromatosis liver is shown with a red arrow pointing to the more dense tissue. ## Lab evaluation - Disorders of iron metabolism are evaluated by: - **Hematocrit and hemoglobin** - **RBC count and 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 indidivuals, 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** ## Lead - 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** - **Diagram**: There is a microscopic image showing red blood cells. There is an arrow pointing to a red blood cell showing basophilic stippling. ## 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. ## 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 notoxic. - **Inorganic salts:** Affects the skin, eyes, GIT, and kidneys. - **Methyl mercury**: Very toxic, highly selective for lipid-rich medium such as brain. ## Molybdenum - Essential trace element: - 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. ## Selenium - Considered as a toxic element in the 1930s, carcinogen in the 1940s, essential element in the 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. - *Anti-oxidant*. - 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 Northern China, North Korea, and Eastern Siberia. - **Toxicity**: hair loss, garlic breath, irritability, mild nerve damage, and nail damage. ## 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, tranferases, 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.** ### Analytical Considerations - 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**. ### Analytical Considerations - 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 1 water). - Reference materials and strict quality control are requisite with each assay run to ensure analytic accuracy. ### Specimen Collection and Processing - 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**. ### Causes of Deficiency - **Disease states** ← insufficient intestinal absorption - **Acquired:** malabsoprtion 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 are formed in the sample 3. **Monochromator** - for the spectral dispersion of the radiation and separation of the analytical line for 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**. ## Lab Methodologies 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.** - **Diagram**: There is a diagram of the ICP-MS instrument showing the flow of the liquid samples starting with a pump and autosampler and ending at the mass spectrometer. The diagram also shows the use of argon gas.