Electromedicina e Instrumentación Biomédica

1. Electromedicina e Instrumentación Biomédica Unidad 4. Moléculas y Biomateriales

2. Contenido 4.1 Moléculas en la Clínica Médica 4.2 Ingeniería de Biomateriales y tejidos 4.3 Temperatura corporal, Grasas y Movimiento

3. Objetivos • Describir los conceptos de Espectrofotometría, Análisis de proteínas, Hematología, etc.; así como los medios para estudiar y medir los niveles de estas sustancias en el cuerpo humano. • Conocer los principales biomateriales, así como los fundamentos de la ingeniería de tejidos. • Describir los principales elementos biosensores empleados, así como sus aplicaciones.

4. Clinical Laboratory Tests

5. Microdialysis • Technique for sampling the chemistry of the individual tissues and organs of the body, and applies to both animal and human studies. • The basic principle is to mimic the function of a capillary blood vessel by perfusing a thin dialysis tube implanted into the tissue with a physiological liquid. • The perfusate is analyzed chemically and reflects the composition of the extracellular fluid with time due to the diffusion of substances back and forth over the membrane. • Microdialysis is thus a technique whereby substances may be both recovered from and supplied to a tissue. • The most important features of microdialysis are: sampling the extracellular fluid, the origin of all blood chemistry; continuously sampling for hours or days without withdrawing blood; and sample purification, simplified chemical analysis by excluding large molecules from the perfusate. However, the latter feature renders the technique unsuitable for sampling large molecules such as proteins. • The technique has been extensively used in the neurosciences to monitor neurotransmitter release, and is now finding application in monitoring the chemistry of peripheral tissues in both animal and human studies.

6. Bilirubin • Red blood cells are replaced approximately every 100 days, which means that every day one percent of the body’s red blood cells, produced in bone marrow, are replaced. Bilirubin is waste resulting from the removal of old red blood cells. • Hemoglobin consists of four subunits. Each subunit has one chain of protein, known as globin, and one molecule of heme. Heme is made up of a single iron atom attached to porphyrin, a ring shaped molecule. When a red blood cell is destroyed, the body recycles the iron. The ring shaped molecule is toxic and consequently broken down into bilirubin. Unconjugated bilirubin is produced in the spleen when the porphyrin is broken down. The unconjugated bilirubin enters the blood stream and travels to the liver where it is converted to conjugated bilirubin and excreted. • Unconjugated bilirubin is produced when red blood cells are destroyed. Abnormally high levels of conjugated bilirubin in the bloodstream, known as jaundice, result from liver disease and can turn skin and the whites of a person’s eyes yellow. Neonatal jaundice, a common problem that occurs after birth, results from the mother’s antibodies attacking the baby’s red blood cells. In general, blood samples can be taken and used to measure bilirubin concentration when diagnosing liver and/or biliary disease. • The most common method to quantitatively measure bilirubin is based on the diazo reaction and spectrophotometry. This technique is used to measure total and conjugated bilirubin in blood serum or plasma.

7. Lactate • Lactate, also known as blood lactate in medicine, is the anionic form of lactic acid present in the blood. Lactic acid is a metabolic intermediate involved in many biochemical processes including glycolysis and gluconeogenesis (the formation of new glucose from noncarbohydrate sources). • Lactate measurement is important because it discovers any disease that causes decreased tissue oxygen consumption. This condition is manifested by increased amounts of lactate in the blood. Some of these diseases include diabetes mellitus, neoplasia (tumor growth), and liver disease. Lactate measurement is important in industry for the regulation and control of food products such as milk. • Lactic acidosis (very high levels of lactate in the blood) is caused by hypoperfusion (a decreased blood flow through an organ). • Prolonged lactic acidosis may result in permanent cellular dysfunction and death. • Lactic acid is measured from a sample of whole blood or plasma. • Lactate is most commonly measured by attaching the enzyme lactate oxidase to a PO2 electrode to detect oxygen consumption, or on a platinum electrode to detect the amount of hydrogen peroxide formed.

8. Creatinine • Found in muscle cells, phosphocreatine converts adenosine diphosphate (ADP) back to adenosine triphosphate (ATP), replenishing the muscle cell’s energy. During this conversion, creatine is produced. • Creatine is usually converted back to phosphocreatine and the cycle starts over. However, when creatine needs to be excreted, it is dehydrated and converted to creatinine. • Creatinine is not reabsorbed when going through the kidneys during urine production. Therefore, it is an ideal candidate measuring the condition of the kidneys. • Measured in blood (serum) and urine, elevated levels of creatinine result from muscle damage or strenuous physical activity. • In a person with kidney disease, creatinine builds up in the blood since it is being produced faster than it is being eliminated. Therefore, measurement of creatinine in blood (serum) is a rough estimate of the health of the kidneys. • Measurement of creatinine from urine is a much better measure of kidney function and is the most prevalent clinical test for approximating glomerular filtration rate (the rate of filtration by the kidneys).

9. Urea • Urea, NH2–CO–NH2, a nitrogen containing molecule, is a metabolic product of breaking down proteins. It is formed in the liver. • Over 90% of urea is excreted through the kidneys to the urine. The body produces urea to rid itself of excess nitrogen. • The liver produces ammonia and converts it to urea as a waste product of gluconeogenesis. Urea is transported in the blood to the kidneys as blood urea nitrogen (BUN). Although the urea nitrogen measurement is often referred to as BUN, it is never measured from whole blood. Urea nitrogen is most often measured from blood serum (watery fluid separated from coagulated blood) and sometimes plasma.An above normal amount of urea in the blood indicates decreased kidney function, and therefore possible kidney disease. • The two primary methods of measuring urea nitrogen are spectrophotometric. The first method measures urea indirectly by quantifying the concentration of the ammonium ion spectrophotometrically.The second method measures urea directly.

10. Glucose • Glucose is the main source of energy for all organisms. Diabetes mellitus is a group of metabolic carbohydrate metabolism disorders in which glucose is underutilized, producing hyperglycemia (high blood sugar levels). The two types of diabetes mellitus are insulin dependent diabetes mellitus (type I) and non-insulin dependent diabetes mellitus (type II). Normally, sugar is not present in the urine, however, when a person has high blood glucose, glucose shows up in the urine. • Insulin is one of the hormones that controls whether glucose is taken out of storage and put into the blood, or vice versa. In type I diabetes, the body does not produce insulin because there is some destruction of the pancreatic islets that produce it. Type II patients produce insulin, but cells do not recognize it (i.e. they have defective insulin receptors). Type I, generally seen in children, is a more severe form of diabetes mellitus, and must be treated with insulin. Type II is seen in older people, and for many, careful control of diet and exercise are adequate treatment. In more severe cases, insulin is taken.

11. Glucosa (2) • Self-monitoring of blood glucose is required for diabetic patients, especially insulin dependent, in order to maintain normal blood glucose levels (glycemia). • When an abnormal amount of glucose is present in the blood, the individual needs to correct the abnormality to avoid short and long term health complications. • By regulating blood glucose levels, patients are mimicking the body by providing themselves with the correct amount of insulin. • Insulin dependent patients need to measure their blood glucose levels about three times a day.

12. Glucosa (3) • One reason blood glucose levels need tight regulation is that glucose is the only source of energy neurons can consume (they do not have the enzymes to consume anything else). • Low blood glucose levels result in hypoglycemia. When this occurs, an individual’s neurons have no source of energy and if low enough, may induce a coma and/or death. • High blood glucose levels result in hyperglycemia. When blood glucose becomes too high, the glucose molecules denature (alter the shape of) proteins, such as collagen and hemoglobin, throughout the body. • Collagen attaches to the lining of blood vessels (the basement membrane), and when glucose denatures collagen, blood vessels are destroyed. This leads to decreased blood flow in the arms and legs (lower perfusion). When glucose denatures proteins associated with neurons, nerve damage occurs and results in a person’s inability to feel. • Diabetes mellitus is the leading cause of amputation because decreased blood flow causes tissue to die and damaged nerves hinder the sensation thus making widespread damage much more likely. • The most common techniques for measuring blood glucose are enzymatic. The glucose oxidase method is a very popular manual procedure used for self-monitoring. The Hexokinase method is widely used in laboratories since the procedures for it are carried out by automated equipment.

13. Amperometric Biosensors for Oxygen and Glucose • Oxygen (PO2):

14. Minerales • Lithium: is not produced in the body nor does it naturally appear in the body. The purpose of lithium measurement is to measure lithium carbonate, which is used in the treatment of the psychiatric disorder manic depression (also known as bipolar disorder). • Sodium and Potassium: are used by the body to maintain concentration gradients in nerve and muscle cells, which allows them to conduct action potentials. To keep these cells working properly, the amount of sodium and potassium in the body must be regulated. It is the responsibility of individual cells to do the regulation. Sodium and potassium enter the body via eating. For people who do not eat properly, it is the kidneys’ job to regulate and compensate the levels of sodium and potassium after eating. For example, if an individual eats a lot of salt, the excess salt in their blood passes in the urine. Potassium chloride injected into the blood stream raises the level of potassium in the body, although it is important to be extremely careful with the dosage. Too much potassium can kill a person by stopping their heart as a result of a decreased concentration gradient, and hence the ability to generate an action potential in the heart muscle cells.

15. Nitrogen by Emission Spectrometry • The concentration of nitrogen, N2, in a sample mixture of gases can be measured by emission spectrometry. • Nitrogen concentration in a mixture of gases is a measurement made in respiratory and pulmonary medicine. • Emission spectrometry involves applying a voltage of 600 to 1500 V dc between two electrodes on opposite sides of an ionization chamber in which the pressure is low (1 to 4 mm Hg or 150 to 550 Pa). • Under these conditions, the nitrogen in the chamber is ionized and emits ultraviolet light (wavelengths from 310 to 480 nm). main components of a nitrogen analyzer:

16. Electrophoresis • technique used to separate charged molecules in a liquid medium with an electric field. • It is extensively used in separation of serum proteins, separation of proteins in urine, determination of molecular weight of proteins, DNA sequencing, genetic disease diagnosis, and comparison of DNA sequences in forensic testing. In an electrophoresis system, charged molecules move through a support medium because of forces exerted by an electric field.

17. Electroforesis This serum protein electrophoresis demonstrates a normal pattern, with the largest peak for albumin. This serum protein electrophoresis demonstrates a decrease in the albumin and an increase in gamma globulins

18. DNA sequencing • There are 20 standard amino acids that make up all proteins. • Each has a carboxyl group and an amino group bonded to an  carbon. • They are distinguished by their different side chains (R groups), and can be separated, identified, and quantified by ion-exchange chromatography. • In high-performance liquid chromatography (HPLC), 0.4 µm spherical cation–exchange resin is packed into a cylindrical column about 5 mm in diameter and 200 mm long. • Amino acids in a buffer solution are pumped through the column at about 13 MPa for about 40 min. • Amino acids with the least positive charge bind weakly to the resin, move most rapidly through the column and elute first to be spectrophotometrically detected at wavelengths less than 300 nm. • Amino acids with a more positive charge bind more tightly to the resin and elute later. • The area under the peak of each amino acid on the chromatogram is proportional to the amount of that amino acid

19. DNA (2) • Amino acids can be covalently joined by peptide bonds to form polypeptides, which generally have molecular weights less than 10,000. • Peptides are obtained by purification from tissue, by genetic engineering, or by direct chemical synthesis. • Within the cell, amino acids are synthesized into a longer polypeptide sequence (a protein) through the translation of information encoded in messenger RNA by an RNA–protein complex called a ribosome. • To separate and purify proteins, cells are broken open to yield a crude extract. Differential centrifugation may yield subcellular fractions. Ion-exchange chromatography can separate proteins by charge in the same way it separates amino acids. Size-exclusion chromatography separates by size. Affinity chromatography separates by binding specificity to a ligand specific for the protein of interest. The purified protein is characterized by ion-exchange chromatography to measure the amount of the protein of interest and the contaminants.

20. Nucleótidos • A nucleotide consists of a nitrogenous base, a pentose sugar, and one or more phosphate groups. • The nucleic acids RNA and DNA are polymers of nucleotides. • The genetic code can be determined by sequencing the four nucleotides that form DNA: A = Adenine, C = Cytosine, G = Guanine, and T = Thymine. A dideoxynucleoside phosphate (ddNTP) analog specific for one of the nucleotides interrupts DNA synthesis to prematurely terminate the fragment at that nucleotide, for example A. • Different analogs terminate C, G, and T fragments. When each of these radiolabeled (with a radioactive compound) fragments is separated electrophoretically, it yields the autoradiogram (by darkening photographic film) pattern in Figure: Nucleotide fragments ending in A, C, G, and T are injected into lanes at the top of electrophoresis columns. The sequence is read from the rows of bands from the bottom up as ACTGTG.

21. DNA sequencing is automated by labeling each of the four fragments with a different colored fluorescent tag. • Then all four fragments are combined and analyzed by a single lane during electrophoresis. • The eluting peaks from successively longer fragments are scanned by a 40 mW UV laser beam, which causes them to fluoresce with one of the four colors. • A computer displays the colored peaks and sequence. • Electrophoresis is carried out in 64 lanes of 200 µm thick acrylamide denatured gel between 300  200 mm optical grade glass for 7 h at 1700 V and 3 mA.

22. Molecules and Biomaterials • A biomaterial is a synthetic material used to replace part of a living system or to function in intimate contact with living tissue. • This definition suggests the development of materials for biological use is accomplished only through an integrated approach of several disciplines: science and engineering, especially material sciences for detailed study and testing of the structural properties and phenomena of materials; biology and physiology, to provide the necessary tools for experimentation based on immunology, anatomy, cellular and molecular biology; clinical sciences, as many medical specialties are directly related to the actual use of biomaterials. • A device fabricated with a biomaterial that is placed inside the living system is called an implant.

23. Types of Biomaterials • Materials for biological use are classified according to their base structure as ceramics, composites, metals, and polymers.

24. Molecules and Tissue Engineering • Tissue engineering can be defined as the application of engineering and life sciences toward the design, growth, and maintenance of living tissues. • This discipline uses living cells and their extracellular products to develop more biological replacements instead of using only inert implants. • Tissue engineering activities can be performed either in vivo (in the body) or in vitro (in solution), the latter being of more interest to bioengineers.

25. Bioartificial tissues • Bioartificial tissues constructed in vitro, are composed of biologic and synthetic substances, and represent a valid alternative to organ transplantation in cases of impaired natural organ function, vital organ failure, or organ donor unavailability. • Although many bioartificial tissues are still in a developmental stage, it is expected that in the future bioartificial organs and tissues will be able to regenerate and perform complex biochemical functions, a behavior that pure artificial implants cannot exhibit that is similar to the natural organs and tissue they will replace.

26. current applications in tissue engineering

27. Regulation of Body Temperature • Body temperature is regulated by a well-designed control system. The average normal body temperature remains almost constant, within ±0.6 C for a healthy person. • It is generally considered to be 37 C when measured orally. When speaking of the body temperature, we usually mean the temperature in the interior—the core temperature. • Deviation of the core temperature beyond the normal range is a signal of a variety of diseases and could have a great impact on a person's life if not treated appropriately. • Temperature is an indicator of the health of a person. While the core temperature is maintained constant, the surface temperature, the temperature of the skin or subcutaneous tissues, varies with the temperature of the surroundings. • This temperature is an important factor when discussing heat loss through sweating (evaporation) from the skin. • Regulation of body temperature uses feedback control, analogous to room temperature control.

28. human temperature regulation system

29. Mecánica • The heat maintenance region is located in the posterior hypothalamus where it combines sensory signals transmitted from the anterior hypothalamus and peripheral temperature sensors to control the temperature-increasing or temperature-decreasing mechanisms of the body. • This part of the hypothalamus acts like a thermostat controller in room temperature regulation. • When the body is too hot, the heat maintenance region triggers temperature-decreasing procedures to reduce body heat by sweating, skin vasodilation, and decreasing tissue metabolism. • Sweating is the main mechanism by which the body causes heat loss via water evaporation. • The effect of skin vasodilation is an increase in heat loss by increasing blood flow to the skin surface, and it is handled through the autonomic nervous system. • Excessive tissue metabolism is inhibited to decrease heat production, which in turn decreases body temperature.

30. Mecánica (2) • On the other hand, temperature-increasing procedures are instituted when the body temperature is too cold. They are: skin vasoconstriction, shivering, and increased thyroxine secretion. • The reaction of skin vasoconstriction, in contrast to skin vasodilation, is caused by stimulation of the posterior hypothalamus. Shivering is a reaction of skeletal muscles to call for an increase in heat production of the body. It is excited by the cold signals from the skin receptors (Guyton and Hall, 1996). The primary motor center for shivering is located at the posterior hypothalamus (dorsomedial portion). The response time of skin vasomotor control and shivering is a matter of minutes. However, the increase of thyroxine secretion is a long-term process of heat production increase through the endocrine system. It usually takes several weeks to complete. Thyroxine is the hormone secreted by the thyroid gland to increase the rate of tissue metabolism. • In addition to the physiological control of body temperature, behavioral response to the change of environment plays an important role in body temperature control, especially in a very cold environment. It is usually an efficient way to maintain our body temperature. For example, we put on more clothes when we expect severe cold weather.

31. Calorimetry • Calorimetry is the measurement of heat given off by a subject. • The term direct calorimetry is used for the direct measurement of heat loss from the subject by radiation, convection, conduction, and evaporation. • Indirect calorimetry measures respiratory gas exchange to infer the amount of heat production. • Heat production is calculated from oxygen (O2) consumption and carbon dioxide (CO2) production and converted to energy expenditure based on an empirical equation. • Calorimetry provides the basis for the science of nutrition, and it is used to estimate nutritional requirements of humans and to evaluate different foods. • It is also a powerful research tool for energy balance and thermal physiology studies. • Finally, it is used clinically as a diagnostic tool for the investigation of metabolic disorders, energy requirements of major illness, and sports medicine.

32. Measurement of Body Fat • Body fat measurement is performed during body composition studies. • It is used in a wide variety of fields, including human biology, medicine, sports science, and nutrition. • Fat measurements may be used to assess obesity in children and adults, and to investigate the effects of malnutrition. Body fat mass is defined as pure fat—triglyceride fat in the body. • Adipose tissue consists of approximately 83% fat, 2% protein, and 15% water. • Fat free mass (FFM) is defined as lean body mass plus nonfat components of adipose tissue. • The assumption used by most body fat assessment methods is that the body is made of two compartments, fat and fat free weight. • The chemical composition of the fat-free body is assumed to be relatively constant. • The fat free body has a density of 1.1 g/cm3 at 37 C with water content of 72 to 74% (typical value 73.2%, Pace and Rathburn, 1945). Body fat, or stored triglyceride, is also assumed to be constant with a density of 0.9 g/cm3 at 37 C. • All body fat methods use either this two–component model or a further division of the fat and fat-free weight into four compartments: water, protein, bone mineral, and fat. • The various body fat measurement techniques may be further divided into two categories. • Some techniques are direct measures of body fat such as deuterium oxide dilution, densitometry, and dual photon absorptiometry (DPA)/dual energy X-ray absorptiometry (DEXA), whereas others are indirect and require cross–validation with other methods. • Examples of indirect methods that will be discussed here are anthropometry and bioelectrical impedance analysis (BIA). • Although MRI and CT are able to directly measure regional fat distribution, especially interabdominal fat content, high cost and long scanning time still limit these two techniques to research studies.

33. Bibliografía • Carr,J.J. y Brown,J.M. “Introduction to Biomedical Equipment Technology” 16. “Medical Laboratory Instrumentation” pp 427-457 • Klein,M. “Analysis of Molecules in Clinical Medicine” CD:/BME310_2003/c3.doc • Monzón,J.E. “Surface Characterization in Biomaterials and Tissue Engineering” CD:/BME310_2003/c4.doc • Wu, Chao-Min. “Body Temperature, Heat, Fat, and Movement” CD:/BME310_2003/c10.doc • Wheeler, L.A. “Clinical Laboratory Instrumentation”; en Webster,J.G(Ed.) “Medical Instrumentation: Application and Design” Ch11, pp 486-517 Complementaria: • Aston,R. “Principles of Biomedical Instrumentation and Measurement” 14. “Clinical Laboratory Equipment”, pp 423-444