The Analysis of Clinical Samples by Atomic Absorption Spectrometry

Transcription

1 Application Note: The Analysis of Clinical Samples by Atomic Absorption Spectrometry Key Words Atomic Absorption Blood Clinical Graphite Furnace Urine Introduction Clinical analysis represents a very important Atomic Absorption applications segment that shares some similarities with environmental analysis. The majority of samples analyzed are taken from the main group of biological fluids, such as whole blood, plasma, serum and urine. To a lesser extent, hard and soft tissues, such as bone, fingernails and hair, may also be used for specific (mainly toxicity) purposes. Nutritional studies involving diet mean that faeces may also be analyzed on occasions. The whole range of atomic absorption techniques can be employed - flame based analysis for the major and minor essential elements, graphite furnace analysis for the trace elements and vapor analysis for the group of toxic, mostly hydride-forming, elements. Sample preparation techniques are often important, especially where the graphite furnace is to be used. The high background levels of the alkali metals means that high background correction efficiency may be required for furnace analyses, together with the ability to use platform cuvettes and matrix modification procedures. Clinical analysis often uses screening techniques to speed up throughput. Rapid methods using STAT flame analysis, for example, can be used to screen out those samples containing normal levels of elements, leaving the abnormal samples to be measured by the longer full analysis procedures. 1) Whole blood and serum: Blood is a highly specialized tissue consisting of several types of cell suspended in a fluid medium containing a variety of dissolved substances that are transported from one part of the body to another. The cellular components consist of red blood cells, which carry respiratory gases, white blood cells, which fight disease, and platelets, which play an important role in the clotting of blood. Blood serum is prepared from whole blood by centrifugation, which separates the fluid from the cellular material. Serum contains many compounds including water, proteins, glucose, lipids, amino acids, salts, enzymes, hormones, antigens, antibodies and urea. Many metallic elements can combine with these compounds. Blood plasma is separated from whole blood by allowing the blood to clot naturally, then separating the fluid from the clot. It is mainly water and inorganic salts, with traces of organic material remaining. 2) Urine: Urine is a pale yellow, slightly acidic fluid excreted by the kidneys and it contains waste products removed from the blood. Urine composition is studied to understand the metabolic processes by the identification and quantification of end products formed and excreted in the urine. You can also obtain evidence that may be useful in the diagnosis and treatment of pathological states by the identification of abnormal substances or substances in abnormal amounts in the urine.

2 3) Analytes: The metallic elements measured in clinical analyses fall into three groups according to their role in human metabolism, and they are usually classed as essential, toxic or therapeutic. Essential elements Essential major elements: Ca, Mg, Na, K Essential minor elements: Zn, Cu, Fe Essential trace elements: Cr, Mn, Mo, Co, V, Se, Ni Essential elements fall into three sub groups of major, minor and trace elements, (shown above) depending on their normal concentration levels in the samples. The elements within each sub group are as follows. Major Essential Elements Calcium Calcium is an essential mineral element comprising up to 2 % of the human body weight. Bones contain about 1 kg in the typical human male but only 1 % of the total calcium content is physiologically active in body fluids. The calcium ion decreases neuromuscular excitability, participates in blood coagulation, activates enzymes and plays an important role in the transfer of inorganic ions across cell membranes. Essentially all the calcium in blood is present in the serum and normal adult serum levels of calcium are in the ranges mmol/l. Magnesium Magnesium ions serve as activators for a number of important enzyme systems engaged in transfer and hydrolysis of phosphate groups. Diagnosis of 'magnesium deficiency tetany' is the most important clinical application in serum, but decreased magnesium levels can be found in cases of the malabsorption syndrome, acute pancreatitis, hypoparathyroidism, chronic alcoholism and delirium tremens, aldosteronism, digitalis intoxication, after protracted I.V. feeding and after excessive loss of magnesium in the urine. Increased serum magnesium levels have been observed in dehydration, severe diabetic acidosis and Addison's disease. Any condition interfering with glomerular filtration results in retention of magnesium, and thus elevation of serum magnesium levels. Normal adult levels are in the range mmol/l. Sodium Sodium is the major cation of extra-cellular fluids. Approximately two-thirds of the 70 g contained in the average adult body is found in these fluids; most of the remainder is in the bone and only a small fraction is located within the cells. Present in the form of a chloride, sodium participates in many vital physiological functions. It is associated with ph control of body fluids, regulation of acid-base reactions, and adjustment of body fluid volume. Sodium can be found in all body tissues and its determination is one of the most frequently performed clinical procedures. Serum level of sodium is relatively constant and extreme fluctuations are used in diagnosis. Low serum sodium levels are found in a variety of conditions, including severe polyuria, metabolic acidosis, Addison's disease, diarrhoea and renal tubular disease. Increased serum sodium levels are found in hyperadrenalism (Cushing's syndrome), severe dehydration due to primary water loss, certain types of brain injury, diabetic coma after therapy with insulin and excessive treatment with sodium salts. The range of normal values for serum sodium are mmol/l. Potassium Potassium is the chief intra-cellular cation found in the human body. Levels in the extra-cellular and intra-cellular fluid are maintained by an active transport mechanism. Low potassium levels can cause excitatory changes in muscle irritability and myocardial function, which are accompanied by characteristic electro-cardiographic changes. Deficiencies of the potassium ion may occur as a result of excessive loss of potassium through prolonged diarrhoea or vomiting. Increased potassium serum levels may be observed in cases of oliguria, anuria, or urinary obstruction. Normal serum potassium levels range from mmol/l. Flame Atomic Absorption Spectrometry (FAAS) is an ideal analytical technique for measuring both major and minor essential elements. FAAS has been used for many years for routine testing of essential elements as it is a quick, simple and accurate technique, which rarely suffers from interference. It can determine most metallic elements at concentrations of low mg/l and even µg/l for some elements. Newer instrumentation can provide enhanced analytical capabilities and increase productivity with complete automation including quality control checking. It is usual to measure major and minor essential elements in serum by flame atomic absorption spectrometry. It is not practicable to aspirate undiluted serum directly into an atomic absorption instrument due to the viscosity of serum, and a dilution factor of at least 1:10 is normally recommended to avoid blockage problems with the nebulizer and/or burner slot.

3 Figure 1: Thermo Scientific ice 3300 AA Spectrometer suitable for the flame analysis of major essential elements Minor Essential Elements Iron Iron was one of the first of the minor elements to be recognised as being necessary for both plants and animals. An average adult male is estimated to contain 4-5 g of iron. Most of the iron exists in complex forms bound to protein either as haeme or porphyrin compounds, especially haemoglobin or myoglobin. Iron in blood is found combined with haemoglobin in the erythrocytes and as transferrin in the plasma in the ratio of approximately 100 to 1. Decreases in serum iron levels are generally due to a deficiency in the total amount of iron present in the body, which may be caused by a lack of sufficient intake or absorption of iron. Low values of serum iron are encountered in hemorragic and hypochromic types of anaemia, in acute and chronic disease, pregnancy, and iron deficiency states. High values of serum iron are observed in hemochromatosis, in forms of anaemia characterized by diminished haemoglobin formation and often in hepatitis. Normal serum iron levels for adult males are in the range mmol/l ( mg/l), while for adult females the range is 8-20 mmol/l ( mg/l). Serum iron levels decrease in the elderly to mmol/l ( mg/l). Zinc Zinc is essential for the growth and propagation of cell cultures and for the functioning of several enzymes. It constitutes about % of the normal human body. Zinc is present in serum, plasma, erythrocytes and leucocytes. It exists in the plasma both as loosely bound and firmly bound fractions associated with serum albumins and globulins. Zinc levels are an important indication of various pathological conditions. The normal values of zinc in serum are in the range of mmol/l ( mg/l). There is little variation with gender or age but there is a diurnal variation with peak concentration occurring about am. Copper Copper is an extremely widespread minor element and can be found in almost all forms of life. A deficiency in copper results in severe derangements in growth and metabolism. The most significant clinical application of copper determination is in its diagnosis of hepatolenticular degeneration (Wilson's disease), in which the total serum copper level is significantly reduced. High blood copper levels occur in most chronic and acute infections, and in leukaemia, Hodgkin's disease, various anaemias, hemochromatosis, rheumatoid arthritis, pregnancy, cirrhosis, and myocardial infarction. Normal levels in serum vary with age and sex. Newborn children are in the range 3-11 mmol/l ( mg/l), children over 6 months and adults mmol/l ( mg/l). Adult males are in the range mmol/l ( mg/l) whereas females are slightly higher, in the range mmol/l ( mg/l). In pregnancy, from 16 weeks to full term, the female values are elevated to mmol/l ( mg/l). Figure 2: STAT Accessory,which may be used for measuring minor essential elements The minor essential elements are present at a concentration that is at the lower end of the normal flame range. They can be determined using the STAT accessory (see Figure 2) or by the graphite furnace technique.

4 Trace Essential Elements Chromium Chromium is considered to be an essential element, but is also known to be toxic when present at elevated concentrations. The metal has been shown to be biologically active in trace amounts, and the chromium-containing molecule known as the 'glucose tolerance factor' is thought to be an insulin co-factor, and hence has been implicated in glucose metabolism. The analysis of blood chromium is used as a reflection of long term exposure, whereas urinary chromium better reflects recent uptake. Normal concentrations of chromium in human whole blood are in the range of mg/l. Manganese Manganese is recognized as an element that is essential to human nutrition. It influences normal growth, and serves an important role in activation of an enzyme (arginase) necessary to the formation of urea. High levels of manganese are considered to be toxic. Symptoms observed in cases of manganese poisoning include psychomotor instability and hallucinations. The rigidity and movements of the limbs are akin to those of Parkinsonism and also suggest a similarity to Wilson's disease. Molybdenum The importance of molybdenum as an essential element has long been established. The molybdenum ion catalyses xanthine and aldehyde oxidase activity. Molybdenum deficiency in man has not been recognized, but instances of both toxicity and deficiency in animals are widely reported. Cobalt Cobalt is recognized as an essential trace element. Cobalt is required in the body for vitamin B12 synthesis. Other important functions of this metal involve the activation of certain enzymes and the formation of haemoglobin. High levels of cobalt induce nausea and vomiting due to the local irritation of the gastric mucosa. Vanadium The normal adult has mg of vanadium mainly in teeth, bones and fat. Most of the ingested vanadium is rapidly excreted in urine; the remainder is retained in the liver and bones. Vanadium is believed to inhibit cholesterol levels 'in vitro'. Vanadium deficiency is reported to cause retarded bone development and impaired reproductive performance. Selenium Selenium was first recognized as an essential element in human metabolism in the 1950's. Deficiencies of selenium are associated with heart disease, muscular dystrophy and reproductive disorders, and the element is an essential component of the mammalian enzyme glutathione peroxidase. As with many essential elements selenium is also known to be toxic when present at higher concentrations. Symptoms of selenium poisoning include abdominal pain and possible partial paralysis. The concentrations of selenium in whole blood reported as normal vary from 50 to 350 mg/l and are dependent on several dietary factors. Figure 3: Thermo Scientific ice 3500 AA Spectrometer enables both flame and graphite furnace analysis to be carried out with automated software changeover Nickel Nickel is considered an essential trace element. The concentration in soft tissue is relatively low, although higher levels are found in the skeleton. It is also reported to be present in RNA and DNA. Nickel deficiency can affect the reproductive process and decrease the 'in vitro' liver oxidation of glycerophosphate. Toxic elements Toxic elements are often defined as those that interfere with metabolic processes. The elements usually included in this group are as follows: Lead, Mercury, Arsenic, Thallium, Cadmium, Aluminium, Boron, Antimony. Lead Lead is a poisonous element, which tends to accumulate in the tissues and bones of exposed individuals. It can be absorbed by inhalation from air or by ingestion of solids or liquids. Most of the lead is excreted in the kidneys, but the process of elimination is much slower than the rate at which it is absorbed. Prolonged exposure to lead may result in anorexia, leadline on gum margin, nausea, severe abdominal pain, paralysis, mental confusion, anaemia, convulsions and brain damage. Mercury Mercury is an environmental contaminant, which has severe toxic implications. Mercury compounds and especially organic mercury compounds, even in low concentrations, are capable of inactivating sulfhydryl bonds and thus interfering with cell function. Alkyel mercury compounds may cause irreversible damage to the central nervous system while inorganic mercury compounds cause reversible damage to tissues such as kidneys and intestines.

5 Arsenic The toxicity of arsenic is well known historically. Arsenic combines readily with proteins due to its great affinity for sulfhydryl groups. This results in the precipitation of proteins, producing gastro-intestinal irritation and irreversible inhibition of important enzyme systems, which are important toxic effects of arsenic. The great affinity of arsenic for tissue proteins is also responsible for the rapid removal of arsenic from the blood. Blood, therefore, is not a good specimen, except in cases in which a large overdose of this substance has been ingested. Thallium Thallium toxicity has been a frequent worldwide occurrence in man and domestic animals. Thallium salts were used as rodenticides and also, both internally and externally, as a depilatory. The characteristic feature of thallium poisoning is loss of hair or occasionally, loss of nails and the skin of the feet. Since this metal is not present in biological material, any amount demonstrated in urine is significant. Cadmium Cadmium is an extremely toxic element. Acute exposure to cadmium leads most commonly to pulmonary oedema. Prolonged exposure may lead to emphysema, proteinuria, and anosmia. Another problem associated with cadmium is its tendency to replace zinc in the body. This can be serious since zinc serves a vital role in the function of many enzymes. Aluminium The determination of aluminium in biological fluids has been the subject of many publications. Most of the attention has been given to the analysis of serum, since serum levels can be used for the diagnosis and monitoring of dialysis patients at risk of aluminium intoxication. In patients with renal failure treated by means of dialysis, it is well established that besides the ingestion of aluminium containing phosphate binders, aluminium contaminated water and dialysis fluids can cause aluminium toxicity. Boron The main cause of human intoxication with boric acid, except for oral ingestion, is resorption through injured or burned skin and mucous membranes. The majority of ingested or resorbed boron is eliminated through the kidneys, however, in cases of boron intoxication it can be detected in blood, spinal fluid, fractions of peritoneal dialysate, brain, liver, spleen and heart tissues. Antimony Antimony and its compounds are extremely toxic. Physical contact, fumes or dust are known to cause dermatitis, keratitis, conjunctivitis and nasal septal ulceration. The volatile hydride derivative of antimony, stibine, is very dangerous and can be responsible for haemolysis and abdominal pains. Therapeutic elements Therapeutic elements are used to treat certain diseases, and their levels in patients undergoing the treatment are monitored to control the dosage and progress of the treatment. Gold Gold complexes are used in the treatment of rheumatoid arthritis and certain other collagen diseases. Radioactive gold has application in treatment of cancer and charged gold leaf has been utilised for neurosurgical procedures as a device for closure of the fragile pia mater. Platinum Platinum salts, especially cisplatin (cisdiamminedichloroplatinium (II)), is a potent anticancer agent with substantial clinical activity against testicular cancer, ovarian cancer, head and neck malignancies, and other tumours. Cisplatin's antitumour effect is mediated through its binding to DNA to form a number of different molecular species and total platinum binding to DNA is directly related to tumour cell kill. Lithium Lithium is widely used in the control of the manic stage of depressive psychosis. Therapeutic levels during the initial treatment are mmol/l of serum, until the threshold of the effectiveness is reached after 6-10 days. The serum level during maintenance therapy is generally kept at mmol/l. Increased lithium levels are toxic, although toxic reactions are rarely seen with serum levels below 1.5 mmol/l. However, they become progressively more severe as the levels increase above 2.0 mmol/l. It is therefore mandatory to regularly monitor serum lithium levels of patients undergoing this treatment. Graphite Furnace Atomic Absorption Spectrometry (GFAAS) is an ideal technique to measure all trace essential elements. Although GFAAS is a slower and more complicated technique than FAAS, it has the distinct advantage of analyzing trace elements at low µg/l and even ng/l for some elements. Another benefit of this technique is that only small sample volumes, of the order of µl, are required. Background (non specific) absorption of the atomic radiation by the sample matrix components can cause large errors, but these can usually be corrected instrumentally, using one or other of the two complementary forms of background correction available, QuadLine (continuum source) or Zeeman effect. Automation of this technique is readily available and is beneficial due to improved measurement and precision. This technique is ideal for the analysis of all trace elements except mercury, for which the sensitivity is poor because of the high volatility of the element. The most frequently used analyses involve one or more of the following procedures: (i) Direct injection into the graphite tube or platform. (ii) Dilution with water, Triton X-100 or dilute acids. (iii) Deproteinisation with stronger acid. (iv) Use of matrix modification.

6 It is rarely necessary to completely digest the sample, unless it is a soft tissue or bone. Chelation/extraction techniques previously used to concentrate the analyte and remove interfering matrix components are seldom required with modern instruments due to their improved performance. Hydride generation AAS is an attractive technique for the hydride forming elements, as the capital cost of the equipment is much less than GFAAS, yet the sensitivities are similar. However, sample digestion is more likely to be required, and precautions have to be taken to ensure that oxidation state interferences are eliminated. 4) Sampling: There are a number of well-defined sampling technologies used for clinical situations. Blood Samples The skin surface is usually cleaned with a disposable alcohol swab before any blood sample is taken. This precaution is particularly necessary in the industrial environment and where a specimen is being taken for analysis of an element such as aluminium, which is subject to a major contamination problem. Capillary blood sampling is best avoided, as it is more subject to contamination. In general, it is normal to collect a venous blood sample through a standard metal disposable needle but for certain analyses, such as manganese, chromium, and nickel, contamination may arise from this procedure. For these elements the blood should be taken through a plastic cannula and the initial 5 ml discarded or reserved for non-trace element analysis. For each element a specific type of container and anticoagulant is recommended. For some trace elements these are significant sources of contamination and selection of the wrong type can completely invalidate the assay. Some examples are listed below: Heparin - May contain zinc, copper and manganese accumulated during chemical separation and purification. Potassium EDTA - Leaches zinc from erythrocytes into plasma. May contain significant amounts of manganese and cobalt. Rubber stoppers/caps - Release significant amounts of zinc. Orange plastic stoppers/caps - Release cadmium. Gel separation systems - Release zinc and other elements. Septum type stoppers/caps - Can also cause lead contamination. To minimize contamination it is best to collect blood in a plain plastic tube and forward the serum. For certain assays, particularly aluminium, polycarbonate tubes are preferred. The separation of serum or plasma from red cells is best done using a clear polythene Pasteur pipette with integral bulb. Glass Pasteur pipettes with rubber teats should never be used because talc or dust inside the teat contains zinc, which can be blown into the pipette during separation thus causing contamination. Release of aluminium from the glass of the pipette can also invalidate assays for this element. Aluminium is so ubiquitous that it is best to send the sample unseparated. For those assays requiring a whole blood specimen, potassium EDTA is the preferred anticoagulant, although some batches may contain significant amounts of manganese and cobalt, as already noted. In order to detect possible contamination, it is generally advisable to send an empty tube with each specimen or batch of specimens forwarded to the laboratory. Urine Samples In general, 24 hour urine samples are collected. The collection is made into a container that has been thoroughly washed with dilute nitric acid (M or 2M) followed by thorough rinsing with de-ionized water. Any 'black rubber' cap seals should be removed prior to washing and use. Polythene containers are not suitable for urine mercury collections and polycarbonate containers should be used. A sterile 'Universal' tube containing 20 ml of the collection is normally forwarded to the laboratory for analysis. Other Samples Biopsy and larger samples of tissue are sectioned with a clean stainless steel scalpel or needle, placed into a dry polycarbonate tube and stored at -20 C until used. Tissues are not stored in formalin as this can cause gross contamination and also leach substantial amounts of cadmium and other elements from the tissue. All specimens that contain, or are suspected to contain, organisms, viruses or substances such as hepatitis B or HIV which are liable to pose a serious infection hazard to laboratory personnel must be clearly identified. 5) Sample preparation: One of the distinct advantages of atomic absorption spectrometry in the analysis of clinical samples is that in most cases very little, or no, sample preparation is required. If sample preparation is required it should involve a minimum of handling and as few reagents as possible. It is essential that all reagents used are of the highest grade available. The difference between 'normal' and 'pathological' levels in biological samples can be quite small; therefore, the danger of contamination must always be borne in mind. It should be remembered that the first requirement for accurate results is cleanliness. Care should be exercised, at all stages, to avoid contamination. The cleaning of all laboratory ware is of prime importance. After use, all glassware should be

7 washed using a phosphate free surfactant detergent, such as Decon 90, and left soaking in 10 % v/v nitric acid until required. 6) Units of measurement: In the typical clinical chemistry laboratory a variety of units are used but the majority of hospital laboratories use the SI molar unit system as the prime unit of reporting. However, for non-clinical users mass units per liter are also quoted. The appropriate relationships are shown below for commonly encountered units. 1 µg/ml = 1 mg/l = 1 part per million (ppm) 1 ng/ml = 1 µg/l = 1 part per billion (ppb) Concentrations of trace elements in urine may be reported per volume, per 24 hour urinary output or per mmol of creatinine or per gram of creatinine. Tissue sample concentrations may be reported as mg or µg per gram or kilogram of wet weight, dry weight or ash weight. 7) Typical results: The following section contains a number of examples of clinical analyses using the various techniques. Appendices 1 and 2 list the reference values for the elements. (a) Simple flame analysis of major elements - using a simple dilution by appropriate factor and addition of La + as a buffer for Ca and Mg determinations or excess Na + as an ionization buffer for K measurements. Element Results mm/l Element Results mm/l Certified Measured Certified Measured Ca Mg Na K Table 2: Analysis of a number of QC sera for the major elements Ca, Mg, Naand K using flame AAS (b) Flame analysis using the STAT accessory for rapid screening of minor elements - uses a simple dilution with deionized water. Sample size may be minimized by using aliquot microsampling. Sample STAT - FAAS QC Scheme Results µmol/l Mean SD µmol/l µmol/l QC QC QC QC QC QC QC QC QC QC QC QC Precipath Precinorm Table 3: Analysis of a number of QC sera for the minor element Cu using flame AAS and the STAT accessory (c) Graphite furnace analysis using the GF95Z Zeeman furnace for Pb determination. 100 ml portions of whole blood are mixed with 900 ml of matrix modifier (which contains 0.1 % v/v HNO3, 0.2 % m/v ammonium dihydrogen phosphate and 0.5 % m/v Triton X-100. Reference Material Certified Value (µg/l) Measured Value (µg/l) NIST SRM 955a ± ± 0.33 NIST SRM 955a ± ± 0.74 NIST SRM 955a ± ± 0.72 NIST SRM 933a ± ± 1.57 Table 4: Analysis of a number of certified reference sera for the trace element Pb using a Zeeman furnace system (d) Vapour analysis using the VP100 hydride accessory - in the example below the sample is acid-digested, treated with concentrated HCl to reduce Se (IV) to Se (III) and determined using 0.1 % m/v sodium borohydride. Sample Se Reference Value (µmol/l) Se Measured Value (µmol/l) Serum ± 10 % 1.02 Serum ± 10 % 0.52 Table 5: Analysis of two certified reference sera for the trace element Se using a VP100 hydride system

8 In addition to these offices, Thermo Fisher Scientific maintains a network of representative organizations throughout the world. The VP100 Vapor/Hydride generation Accessory 8) Conclusions: Atomic Absorption is a technique that is ideally suited to the analysis of clinical samples. Using the range of accessories, (flame, furnace and vapor) it is possible to analyze major, minor and trace level concentrations of all the clinically significant elements in a wide range of sample types. Modern AAS systems have the detection power and are easy to use, with Wizard-driven software to guide the user through method setup and analysis with the minimum of effort. Africa Australia Austria Belgium Canada China Denmark Europe-Other France Germany India Italy Japan Latin America Middle East Netherlands South Africa Spain Sweden/Norway/ Finland Switzerland UK USA Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. FM Thermo Electron Manufacturing Ltd (Cambridge) is ISO Certified. AN40835_E 03/08C Part of Thermo Fisher Scientific