Extraction Techniques and Applications: Biological/Medical and Environmental/Forensics

K. Lew , in Comprehensive Sampling and Sample Preparation, 2012

3.05.5.3 Tourniquet

A tourniquet allows for pressure to be applied to the arm so that venous blood returning to the heart can be slowed down. As a result, the blood vessel walls become temporarily occluded and the veins distend due to the pooling of blood. This allows veins to become more visible and easier to palpate.

Some tourniquets are made out of Velcro (Figure 12) but many tourniquets used are made out of a stretchy material (more tourniquets are now being produced without latex due to the increasing number of allergies). In a healthcare setting, these stretchable tourniquets are usually meant for a single use. A blood pressure cuff can also be used as a tourniquet, which is commonly used when a large volume of blood is collected (such as for transfusion purposes).

Figure 12. Tourniquets. Tourniquets help to distend the veins for phlebotomy. They can be a latex-based or a latex free strap, or contain a velcro closure. A blood pressure cuff is also suitable for slowing the flow of venous blood.

The phlebotomist should not leave the tourniquet on the patient's arm for longer than a minute. This increased pressure against the vessel walls allows plasma and small molecules to flow through capillary walls and into the tissue. This process is known as hemoconcentration; it results in a relative increase in the number of red blood cells as well as higher-molecular-weight compounds in the sample drawn. With prolonged tourniquet application time, test results such as albumin, cholesterol, coagulation proteins, and red cell count are falsely increased (Section 3.05.8.4).

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123813732000685

Marine Envenomations

Pratap Chand , in Clinical Neurotoxicology, 2009

Clinical Management of Stonefish Stings

Immediate first aid treatment requires immobilization of venom at the penetration site by firm constrictive bandaging or by managed tourniquet sited between wound and proximal flexure. Prehospital care should address recognition of the injury as a potential envenomation, gentle removal of visible spines, direct pressure to control bleeding, administration of analgesia, and transport for definitive medical evaluation. Recognition of serious systemic symptoms and prompt attention to airway, breathing, circulation, cardiopulmonary resuscitation, and treatment for anaphylaxis are lifesaving. 53 Hot water immersion is widely recommended as effective initial treatment for envenomations after removal of visible spines and sheath to inactivate the thermolabile components of the venom. The affected limb should be immersed in water no warmer than 114°F, or 45°C, with care not to inflict thermal burns. Emergency management of stonefish envenomations includes prompt analgesia, wound management, antivenom administration, and supportive treatment for significant envenomations. 54 Wound debridement and surgical removal of embedded spines are indicated when they are in proximity to joints, nerves, or vessels because retained spines continue to envenomate. Retained fragments also act as foreign bodies, causing inflammation and later granuloma formation and leading to delayed healing and secondary infection. Adjunctive regional or local anesthesia offers reliable, prompt, and prolonged analgesia, allowing simultaneous debridement of the wound. Parenteral analgesics and or sedatives may be needed for patients with wounds that are difficult to immerse or anesthetize or for people exhibiting significant anxiety reactions to the envenomation. Tetanus prophylaxis is indicated in all patients with insufficient immunization histories. Stonefish antivenom from Australia's CSL is recommended only for predilution intramuscular usage. In serious envenomations, a slow intravenous administration of antivenom diluted in 50 to 100 mL of isotonic sodium chloride solution and run through at least 20 minutes is preferable. 54 This is a hyperimmunized equine antisera, and there are risks of allergic reaction and serum sickness in the recipient. Skin testing and or pretreatment with subcutaneous epinephrine and an intramuscular antihistamine, adding an intramuscular corticosteroid for known hypersensitivity, should precede administration. 54

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780323052603500484

Snakes

S.L. Thornton , in Encyclopedia of Toxicology (Third Edition), 2014

Clinical Management

The management of venomous snakebites is based on supportive care and appropriate timely antivenom therapy. Supportive care such as local wound care and pain treatment are important but potentially harmful interventions such as tourniquets, cutting, suction, and application of electricity should be avoided. Antivenom is the definitive treatment. Antivenom is produced by injecting a host animal, such as a horse or sheep, with diluted venom. The host animal then mounts an immune response to the venom and IgG are produced. The IgG are harvested and purified. In some cases the IgG are further treated with enzymes, which create Fab fragments. After further purification, these Fab fragments have the advantage of being much less antigenic than whole IgG preparations. Hypersensitivity reactions are a concern with the administration of any antivenom, especially equine-derived whole IgG products. These reactions can range from urticaria to life-threatening anaphylaxis. When using whole IgG antivenom, skin testing prior to administration is recommended. However, hypersensitivity reactions can still occur with a normal skin test result and standard anaphylaxis therapy (epinephrine, antihistamines, corticosteroids) should be available. Antivenom can be monovalent and directed toward only one species of snake or polyvalent and directed toward multiple snake species. Most of the antivenoms produced for use in the developing world are polyvalent. Some examples include the South African Institute for Medical Research Polyvent Snake Venom, which is directed against 10 different snake species or FAV-Afrique, which is made from venom of 11 different snake species. Additionally, antivenom frequently shows a high degree of cross reactivity. For instance, in North America the Crotalidae polyvalent immune Fab (ovine) antivenom is made from the venom of four pit viper species yet is effective in neutralizing the venom from over a dozen North American pit vipers species. The exact dose of antivenom will depend on the specific product and the clinical picture. Typically antivenom is administered until clinical signs of envenomation are controlled. It is important to administer antivenom as quickly as possible. Antivenom is effective at halting further damage from venom but does not reverse preexisting venom effects. The availability of antivenom will vary from country to country. The WHO website has an extensive searchable database of all medically important venomous snakes and their corresponding antivenom.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123864543007867

Snake, Crotalinae

Gary W. Everson , in Encyclopedia of Toxicology (Second Edition), 2005

Clinical Management

Most first-aid measures that have been historically employed are of little value and some are dangerous and worsen medical outcome. The use of ice to prevent the spread of venom has been linked to an increased frequency of limb amputations and should never be employed. The incision of fang marks to relieve venom is ineffective and can result in nerve or artery damage. Tourniquet use may impede blood flow in the affected limb and contribute to local tissue damage. The application of electric shock at the bite site has shown to be ineffective in clinical trials and is also dangerous. However, the use of a properly applied constriction band as opposed to a tourniquet may possibly be effective in slowing the lymphatic distribution of venom. Anyone bitten by an unidentified snake requires evaluation in an emergency facility equipped to provide basic and advanced clinical life support. Aggressive supportive care is at least as important as the proper administration of antivenom in the outcome of a patient bitten by a venomous snake.

It is important to evaluate the clinical presentation of the patient as well as laboratory data to determine and guide the administration of antivenom. However, antivenom is not required in all patients who are envenomated and may not be necessary if there is no significant tissue swelling, systemic symptoms are absent, and laboratory parameters are normal.

When symptoms develop, a decision to start antivenom should be made. Patient response to the antivenom must be evaluated at frequent time intervals following administration of the initial dose to determine if further antivenom is required. CroFab™, a polyvalent sheep-derived (ovine) antivenom produced by Protherics, was approved by the Food and Drug Administration and is preferred over the older equine-based, Antivenin (Crotalidae) Polyvalent™ (Wyeth), because it is less antigenic and is now distributed much more widely than the Wyeth product. CroFab™ is marketed by Savage Laboratories. CroFab™ consists of highly purified ovine Fab fragments capable of neutralizing the toxic effects of most North American Crotalinae venoms. It contains a mixture of venom-specific Fab fragments and contains few of the proteins responsible for the allergic reactions associated with the Antivenin (Crotalinae) Polyvalent™ (Wyeth). Clinical trials to date have shown CroFab™ to be effective and safe in the treatment of Crotalinae envenomations. Acute reactions in the preliminary studies were minimal and serum sickness was virtually nonexistent. The initial dose of CroFab™ is four to six vials. Further doses may be given if symptoms continue to progress. Patients exhibiting life-threatening symptoms may require 30 or more vials of antivenom. The effectiveness of antivenom is directly related to its timely administration and the provision of adequate dosing. Clearly, the effectiveness of antivenom decreases as administration time is delayed.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B0123694000008826

Volume 1

Chi-Hsien Chen , Yue L. Guo , in Encyclopedia of Environmental Health (Second Edition), 2019

Latex

Natural rubber latex (NRL) is the product derived from the milky fluid produced by the tropical rubber tree, which secretes latex to seal and protect the wounded sites. NRL is widely used in the production of everyday articles, including household gloves, toys, balloons, condoms, baby pacifiers, sports equipment, elastic straps, mattresses, tires, and adhesives. Many medical devices also use NRL as material, such as gloves, catheters, drainage tubes, anesthetic masks, and tourniquets.

NRL can cause a wide spectrum of allergic reactions, ranging from urticaria, rhinoconjunctivitis, and asthma to extensive angioedema and life-threatening anaphylaxis. Sensitization and allergic reaction to NRL can occur from direct contact of NRL material with the skin, and mucosal and serosal membranes.

Latex-induced asthma usually results from inhalation of airborne NRL allergens bound to powder particles of gloves. Health-care workers wearing NRL-free gloves may be exposed to disseminated airborne NRL allergens from NRL gloves used by coworkers. Other occupations at risk of respiratory allergy to NRL are usually NRL glove users, such as hairdressers, food processors, pharmaceutical workers, and laboratory workers. In health-care workers, NRL-associated proteins Hev b 5 (acidic protein), Hev b 6 (hevein), and Hev b 7 (patatin) are the most common sensitizers identified by skin prick test. Among individuals sensitized to NRL, about a half have cross-reactions with fruits, such as banana, mango, kiwi fruit, walnut, and avocado. The cross-reactions can induce latex-fruit syndrome with symptoms ranging from itching of the throat to oral or facial swelling, rhinoconjunctivitis, and anaphylactic shock. Reduction of NRL exposure in health-care institutions by using NRL-free gloves can prevent NRL allergy in health-care workers.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780124095489114198

Extraction Techniques and Applications: Biological/Medical and Environmental/Forensics

J.W. Guthrie , in Comprehensive Sampling and Sample Preparation, 2012

3.01.4.2 Blood

Blood is the most common sample type used in laboratory diagnostics since it is easily accessible and can provide a wealth of information on the physiological and biochemical state of an individual, such as disease, mineral content, drug effectiveness, and organ function. Aspects that should be considered for blood collection are the type of sample (venipuncture, arterial puncture, and skin puncture), the site of collection and its preparation, the position and health of the patient, the tourniquet technique, the needle bore size, and the type of collection tube.

Venipuncture via the median cubital vein is the preferred location since it is easily accessible and results in the least amount of discomfort and stress for the patient. Blood taken from fragile veins can result in the leakage of hemoglobin, and other intracellular erythrocyte components into the blood sample, which can interfere with a variety of analytical reactions, cause increases in absorbance at 415 and 540   nm, and can be detected on PAGE, MALDI, and SELDI. 2 Factors such as time elapsed since venipuncture before separation of plasma or serum from cells and processing temperature may also have a significant impact on the results. 43,61

To prepare the collection site for puncture, alcohol must first be applied to sterilize the site. The alcohol must be allowed to evaporate before puncture to prevent residual alcohol from mixing with the sample, which can result in hemolysis and increased levels of certain analytes. The posture of the patient can alter the concentration of plasma proteins by as much as 10% due to variations in blood volumes when standing versus sitting or lying down. Improper tourniquet technique, such as duration, can lead to increased concentrations of proteins such as fibrinogen, potassium, and lactic acid.

Hemolysis can affect the specimen in a number of ways that may affect downstream analysis. Hemolysis changes the intracellular and extracellular concentrations of components such as lactic dehydrogenase, alkaline phosphatase, haptoglobin, bilirubin, potassium, and phosphorus within the specimen. This may result in changes in the concentration of the analyte if it was one of the components also present within the hemolyzed blood cells or if the cellular components dilute the sample. Colored compounds within the cell may considerably increase the low absorbance wavelength range (300–500   nm), which can affect the reported concentrations of colored analytes. The pH of the specimen may also be altered, which can interfere with chemical measurements such as enzyme activity kinetics. Hemolysis and clotting due to problems drawing blood from a patient have been reported to account for approximately 80% of the errors that occur in the sampling phase. 62

Factors such as needle bore size, type of collection tube, and centrifugation speed and duration must be carefully controlled to prevent hemolysis. 53,63 These factors may not be the same for all patients. For example, the bore size of the needle may depend on the disease state of the patient, since certain patient conditions may cause the blood to undergo hemolysis more easily than a normal healthy patient. 52 Without due care, the blood sample may be contaminated.

One of the issues to be aware of in the process of blood collection is that, during coagulation, the cellular components, especially platelets, can secrete a variety of compounds into the plasma. Removal of cellular components must be done immediately after collection to minimize these effects. This can be accomplished by filtration through a 0.2-μm membrane filter, double centrifugation of the sample, or use of additives to minimize platelet activation such as a mixture of citrate, theophylline, adenosine, and dipyridamole. 8

The storage of whole blood at cooler temperatures has its own associated problems. Low temperatures are generally required to preserve sample proteins; however, erythrocytes are less stable at lower temperatures, and storage of blood may result in hemolysis and the release of intracellular components into the sample. 2

The tube used for collection and the additives within the tube will depend on the next step in the preparation of blood, which is the separation into serum or plasma. The separation of blood into plasma or serum has considerable implications for proteomic research.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B978012381373200065X

Clinical Epidemiology

David L. Sackett , R. Brian Haynes , in International Encyclopedia of Public Health (Second Edition), 2017

Typical Clinical Epidemiologic Investigations

Typical clinical epidemiological investigations include:

1.

Comparing patients' symptoms and signs with the results of "reference standard" diagnostic tests. High quality studies of this sort sample patients in whom it is clinically sensible to suspect a specific diagnosis (e.g., patients suspected of chronic airflow limitation), carry out specific bits of the clinical examination (e.g., the position of the thyroid cartilage relative to the suprasternal notch), and then carry out an independent, "reference standard" test (e.g., spirometry) while "blind" to the results of the clinical examination. The results of these studies, commonly expressed as likelihood ratios, improve both the accuracy and efficiency of the clinical examination. In doing so, they have confirmed the usefulness of some traditional signs and symptoms (e.g., the presence of an S3 gallop on cardiac auscultation of a patient with suspected heart failure), added new signs and symptoms (e.g., clinical prediction rules for deep vein thrombosis and the "Ottawa ankle rule" for ruling out the need for ankle radiographs) and, equally important, shown that other signs and symptoms are useless (e.g., the tourniquet test for carpal tunnel syndrome).

2.

Relating patients' later outcomes ("prognoses") to the results of earlier ("baseline") clinical and laboratory findings. High quality studies of this sort sample patients at the very start of their illness (an "inception cohort"), perform baseline clinical and laboratory examinations on them, and then follow them to the conclusion of their disease. The results of these studies, often referred to now as "clinical prediction guides", provide more accurate predictions and advice to patients at the start of an illness.

3.

Randomized clinical trials in which consenting patients are assigned, by a system analogous to tossing a coin, to receive or not receive a new ("experimental") treatment and then closely followed for the occurrence or prevention of unfavourable outcomes. High quality studies of this sort have been applied to medications (e.g., caffeine for premature babies), operations (e.g., carotid endarterectomy for threatened stroke), behavioral and educational interventions (e.g., behavioral maneuvers for improving compliance with medications and exercise), health professionals (e.g., the nurse practitioner), and the like. The results of these studies determine whether new treatments or other health care interventions do more good than harm. For example, the preventive and therapeutic interventions for coronary heart disease that have been validated in randomized trials are credited with halving both the incidence and case-fatality of myocardial infarction in high-income countries.

4.

Cluster randomized trials in which groups of patients, clinicians, clinics, hospitals or communities are randomly allocated to receive or not receive an intervention to improve the application of validated health interventions or services. This approach has become the mainstay of knowledge translation research and implementation science and is used, for example, to assess the value of computerized clinical decision support, continuing education, and quality improvement interventions.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780128036785000771

Anesthetics

F Liu , ... T.A. Patterson , in Encyclopedia of Toxicology (Third Edition), 2014

Local Anesthetic Agents

Local anesthetic agents can cause reversible local anesthesia by inducing the absence of pain sensation without affecting consciousness. All local anesthetics consist of three components: an aromatic portion, an intermediate chain, and an amine group. The intermediate chain, which connects the aromatic and amine portions, is composed of either an ester or an amide linkage. Thus, the local anesthetics are further classified into two categories: esters and amides. Anesthetics such as benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine/larocaine, piperocaine, propoxycaine, procaine/novocaine, proparacaine, and tetracaine are esters. Examples of amides include articaine, bupivacaine, cinchocaine/dibucaine, etidocaine, levobupivacaine, lidocaine/lignocaine, mepivacaine, prilocaine, ropivacaine, and trimecaine. Both classes of local anesthetics act mainly by preventing sodium influx through voltage-gated sodium channels in the neuronal cell membrane into the cytoplasm, thus preventing the local membrane from depolarization. Therefore, an action potential cannot be generated and spread; the signal conduction is inhibited, thereby effecting local anesthesia. In general, all nerve fibers are sensitive to local anesthetics. However, nerve fibers that have a smaller diameter are more readily blocked than those with larger diameter. For instance, the pain sensation, transmitted by small and unmyelinated nerves, can be blocked more rapidly than other sensations. In addition, local anesthetics have a greater binding affinity to sodium channels, which are in an activated state. An active neuron is more sensitive to local anesthetics. This is referred to as state-dependent blockade.

Lipid solubility of anesthetics is the most important factor in determining the intrinsic anesthetic potency, which is influenced by the aromatic portion of the molecule. The duration of anesthesia is determined by the extent of local anesthetics binding to proteins, which are immersed in lipids of the membrane. The greater the binding affinity to nerve proteins the longer the anesthetic activity will persist.

Uses and Exposure Routes and Pathways

Local anesthetic agents are administered to the areas around the nerves to be blocked (skin, subcutaneous tissues, intrathecal, and epidural spaces). Their activities vary considerably. Topical anesthesia is the administration of local anesthetics to the skin or other body surface. Most anesthetics are barely absorbed through intact skin, and the effectiveness of anesthesia is affected. Eutectic mixtures, such as 2.5% lidocaine and 2.5% prilocaine (EMLA), improve the effectiveness of the anesthetic on intact skin by lowering the melting temperature of the mixture compared with that of each individual anesthetic.

Infiltration anesthesia is the injection of local anesthetics into the tissue to be anesthetized. Amide anesthetics with a moderate duration of action are commonly used (i.e., lignocaine, prilocaine, and mepivacaine) to cause infiltration anesthesia for minor surgical procedures.

Epidural anesthesia is the administration of local anesthetics to the epidural space between the dura mater and the periosteum lining the vertebral canal. The conduction is blocked at the intradural spinal roots and the absence of pain sensation can be achieved. Spinal anesthesia is the application of local anesthetics into the cerebrospinal fluid at the site of the lumbar spine.

Intravenous local anesthesia is the injecting of local anesthetics into a vein of a limb (a leg, foot, or lower arm, hand) that has been exsanguinated and blocked by a tourniquet. The anesthesia is limited to the area that is excluded from blood circulation. One restriction that should be kept in mind is that bupivacaine and etidocaine should never be used for intravenous local anesthesia due to the risk of cardiotoxicity.

Toxicokinetics

All local anesthetics are weakly alkaline. They exist in both nonionized and ionized forms. The term pK a of a weak base is defined as the pH at which both forms exist in equal amounts. At physiological pH (7.4), local anesthetics are more ionized than nonionized (as their pK a values are >7.4). However, the proportions vary among the anesthetics. Nonionized anesthetics can pass through the lipid cell membrane more rapidly than ionized ones. Therefore, an anesthetic agent that has a higher proportion of nonionized form will reach the target site more quickly and will have a faster onset of blocking.

Ester and amide anesthetics are metabolized through different routes. The metabolism of esters (except cocaine) is through hydrolysis in plasma by the enzyme pseudocholinesterase and they have a short half-life. Cocaine is hydrolyzed in the liver. Ester metabolite excretion is through the kidney. The amides undergo enzymatic degradation by microsomal enzymes located in the liver. This is a slower process, hence the half-life of amides is longer and they can accumulate if given repeatedly.

Acute and Short-Term Toxicity of Local Anesthetics

Allergic reactions to local anesthetics are rare. Esters produce the most anesthetic-induced allergic reactions due to their metabolite, para-aminobenzoic acid (PABA), a well-known allergen. Hypersensitivity to amide local anesthetics is seldom observed. Since there is no cross-allergy between esters and amides, amides can be used as alternatives in patients who show hypersensitivity to esters. Therefore, amides are now more commonly used than esters.

Local anesthetics may be toxic if sufficient amounts are absorbed into the systemic circulation or administered improperly. The toxicity can be at local and systemic levels. The local adverse effects of anesthetics may include prolonged anesthesia and paresthesias, which may become irreversible.

Systemic toxicological effects of local anesthetics involve the central nervous, cardiovascular, and immune systems. In general, the CNS is more sensitive to local anesthetics than the cardiovascular and immune systems. Therefore, symptoms and signs of CNS disturbances usually occur earlier, showing excitatory effects in the brain before the depressant effects. Myocardial depression and bradycardia indicate the effects of local anesthetics on the cardiovascular system. On very rare occasions (<1%), immunoglobulin E (IgE)–mediated allergic reactions can occur.

CNS toxicity is usually related to the intrinsic potency of the anesthetics. Procaine is least potent and least toxic following a rapid intravenous injection. Bupivacaine, tetracaine, and etidocaine are the most potent compounds in terms of intrinsic anesthetic and CNS convulsive activity. Lidocaine, mepivacaine, and prilocaine are intermediate in anesthetic potency and convulsive activity.

Chronic Toxicity (Animal/Human)

Local anesthetics can easily cross the placenta by passive diffusion. There is now general agreement that properly conducted epidural anesthesia does not cause neurobehavioral changes in the newborn.

Carcinogenesis

Long-term studies in animals to evaluate carcinogenic potential have not been conducted on most local anesthetics. A minor metabolite of lidocaine, 2,6-xylidine, has been found to be carcinogenic in rats.

Mutagenesis and Impairment of Fertility

Mutagenic potential or the effects on fertility of most local anesthetics has not been reported, although animal experiments have reported decreased pup survival in rats and an embryocidal effect in rabbits when bupivacaine hydrochloride was administered to these species in doses comparable to 9 and 5 times, respectively, the maximum recommended daily human dose. However, there are no adequate and well-controlled studies in pregnant women of the effects of the same agent on the developing fetus.

Clinical Management

Administration of local anesthetic agents should be stopped if a patient shows any signs or symptoms of toxicity during anesthesia. An intravenous lipid emulsion treatment, called lipid rescue, has been shown to be effective in treating cardiotoxicity based on animal evidence and human case reports. The use of lipid rescue has been encouraged in the United Kingdom and officially promoted as a treatment by the Association of Anesthetists of Great Britain and Ireland.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123864543000038

Snakebites in Hungary—Epidemiological and clinical aspects over the past 36 years

Tamás Malina László Krecsák Zoltán Korsós Zoltán Takács , in Toxicon, 2008

First-aid methods, such as incision, wound washing with KMnO4 or H2O2 solutions, and tourniquet use, have long been contraindicated ( Warrell, 1993) but were still applied in several cases. These methods are delaying expedite transport to hospitals and could easily be a contributing factor to some of the extensive local tissue damage we observed. For snake-handlers in Hungary, in conjunction with the accepted and established first-aid measures (e.g., Warrell, 2005b), we advocate seeking immediate medical care after any venomous snakebite while supporting vital functions as priority. Among our patient population, a mobile phone seems to be one of the best "first-aid" responses.

Read full article

URL:

https://www.sciencedirect.com/science/article/pii/S0041010107004473

Kinetics of zinc status and zinc deficiency in Berardinelli-Seip syndrome

Maria Goretti do Nascimento Santos , ... José Brandão-Neto , in Journal of Trace Elements in Medicine and Biology, 2012

Biochemical analysis

All procedures regarding manipulation of zinc samples were performed according to international standards. Venipuncture was performed using plastic syringes without a tourniquet. All tubes and pipette tips were trace metal-free. All material used for zinc collection, separation, and storage was plastic trace metal-free, and procedures were performed according to guidelines for trace elements [16]. Blood samples, immediately after collection, were maintained in trace metal-free tubes without anticoagulants and kept 120   min in the stainless steel incubator (FANEM 502, São Paulo, Brazil) until clot formation. Next, 500   μL of serum were collected with plastic trace metal-free pipettes and transferred to plastic tubes containing 2000   μL of ultra pure water (Milli-Q plus, Millipore, USA) to dilute the serum. Samples showing hemolysis were discarded because erythrocytes are rich in zinc [17]. Urine was collected in a plastic trace metal-free recipient, the volume was measured in a cylinder and then 500   μL was collected and diluted with 2000   μL of ultra pure water. Serum and urine samples were frozen and stored at −20   °C, for up to 2 months until analysis. Serum and urine zinc samples from each individual were analyzed in duplicate within the same assay using an atomic absorption spectrophotometer (SpectrAA-200, Varian, Victoria, Australia) in accordance with the manufacturer's instructions. Assay sensitivity was 0.01   μg/mL, the intra-assay coefficient of variation was 2.6%, and the normal reference values were 0.7–1.20   μg/mL. Zinc concentration of the samples was determined using a standard solution from our laboratory as quality control, in order to check reproducibility and accuracy of the measurements. The standard zinc solution (0.5   μg/mL) was obtained by diluting the stock zinc solution (500   μg/mL) prepared from 0.5   g of zinc powder purchased from Merck (Darmstadt, Germany) and dissolved in a small volume of hydrochloric acid (HCl, Merck, Darmstadt, Germany), which was later reconstituted to 1   L, with 1% HCl (v/v). Zinc concentration of the samples was determined with a widely used standard solution from our laboratory as quality control [11]. Wavelength was 213.9, lamp current was 5   mA and all other procedures, such as calibrations and measurements were carried out in accordance with the manufacturer's instructions.

Clinical laboratory parameters were also evaluated at the beginning of the study and after 3 months using standard clinical laboratory methods: hematologic (Horiba ABX Diagnostics, Micros 60, Montpellier, France), biochemical (Dade Behring, Dimension AR, IL, USA). The analyses were performed by the Multidisciplinary Laboratory of Chronic Degenerative Diseases.

Read full article

URL:

https://www.sciencedirect.com/science/article/pii/S0946672X11002690