Chemistry of the blood group substances : The exact chemical structure of some blood groups has been identified,

as have the gene products (i.e., those molecules synthesized as a result of an inherited genetic code on a gene of a chromosome) that assist in synthesizing the antigens on the red cell surface that determine the blood type. Blood group antigens are present on glycolipid and glycoprotein molecules of the red cell membrane. The carbohydrate chains of the membrane glycolipids are oriented toward the external surface of the red cell membrane and carry antigens of the ABO, Hh, Ii, and P systems. Glycoproteins, which traverse the red cell membrane, have a polypeptide backbone to which carbohydrates are attached. An abundant glycoprotein, band 3, contains ABO, Hh, and Ii antigens. Another integral membrane glycoprotein, glycophorin A, contains large numbers of sialic acid molecules and MN blood group structures; another, glycophorin B, contains Ss and U antigens. The genes responsible for inheritance of ABH and Lewis antigens are glycosyltransferases (a group of enzymes that catalyze the addition of specific sugar residues to the core precursor substance). For example, the H gene codes for the production of a specific glycosyltransferase that adds l-fucose to a core precursor substance, resulting in the H antigen; the Le gene codes for the production of a specific glycosyltransferase that adds l-fucose to the same core precursor substance, but in a different place, forming the Lewis antigen; the A gene adds N-acetyl-d-galactosamine (H must be present), forming the A antigen; and the B gene adds d-galactose (H must be present), forming the B antigen. The P system is analogous to the ABH and Lewis blood groups in the sense that the P antigens are built by the addition of sugars to precursor globoside and paragloboside glycolipids, and the genes responsible born June 14, 1868, Vienna, Austrian Empire [Austria] died June 26, 1943, New York, N.Y., U.S.

Austrian American immunologist and pathologist who received the 1930 Nobel Prize for Physiology or Medicine for his discovery of the major blood groups and the development of the ABO system of blood typing that has made blood transfusion a routine medical practice.

After receiving his M.D. in 1891 from the University of Vienna, Landsteiner studied organic chemistry with many notable scientists in Europe, including the German chemist Emil Fischer. In 1897 he returned to the University of Vienna, where he pursued his interest in the emerging field of immunology and in 1901 published his discovery of the human ABO blood group system. At that time, although it was known that the mixing of blood from two individuals could result in clumping, or agglutination, of red blood cells, the underlying mechanism of this phenomenon was not understood. Landsteiner discovered the cause of agglutination to be an immunological reaction that occurs when antibodies are produced by the host against donated blood cells. This immune response is elicited because blood from different individuals may vary with respect to certain antigens located on the surface of red blood cells. Landsteiner identified three such antigens, which he labeled A, B, and C (later changed to O). A fourth blood type, later named AB, was identified the following year. He found that if a person with one blood typeA, for examplereceives blood from an individual of a different blood type, such as B, the hosts immune system will not recognize the B antigens on the donor blood cells and thus will consider them to be foreign and dangerous, as it would regard an infectious microorganism. To defend the body from this perceived threat, the hosts immune system will produce antibodies Blood Chemistry Definitions : Hematology HEMATOCRIT (HCT) The word hematocrit means "to separate blood", a procedure which is done following the blood draw through the proper use of a centrifuge. Hematocrit is the measurement of the percentage of red blood cells in whole blood. It is an important determinant of anemia (decreased), polycythemia (increased), dehydration (elevated), increased R.B.C. breakdown in the spleen (decreased), or possible overhydration (decreased). Normal Adult Female Range: 37 - 47 % Optimal Adult Female Value: 42 % Normal Adult Male Range: 40 - 54 % Optimal Adult Male Value: 47 % Normal Newborn Range: 50 - 62 % Optimal Newborn Value: 56 % Panels: Hematology

HEMOGLOBIN (HGB) Hemoglobin is the main transport of oxygen and carbon dioxide in the blood. It is composed of globin a group of amino acids that form a protein and heme which contains iron atoms and imparts the red color to hemoglobin. As with Hematocrit, it is an important determinant of anemia (decreased), dehydration (increased), polycythemia (increased), poor diet/nutrition, or possibly a malabsorption problem. Normal Adult Female Range: 12 - 16 % Optimal Adult Female Value: 14 % Normal Adult Male Range: 14 - 18 % Optimal Adult Male Value: 16 % Normal Newborn Range: 14 - 20 % Optimal Newborn Value: 17 % Panels: Hematology MCH (Mean Corpuscular Hemoglobin) Hemoglobin x 10 R.B.C. Mean Corpuscular Hemoglobin (MCH) gives the average weight of hemoglobin in the red blood cell. Due to its use of red blood cells in its calculation, MCH is not as accurate as MCHC in its diagnosis of severe anemias. Decreased MCH is associated with microcytic anemia and increased MCH is associated with macrocytic anemia. Normal Adult Range: 27 - 33 pg Optimal Adult Value: 30 pg Panels: Hematology MCV (Mean Corpuscular Volume) Hematocrit x 10 R.B.C. The Mean Corpuscular Volume reflects the size of red blood cells by expressing the volume occupied by a single red blood cell. Increased values may indicate macrocytic anemia or B6 or Folic Acid deficiency and decreased values may indicate microcytic anemia, possibly caused by iron deficiency.

Normal Adult Range: 80 - 100 fL Optimal Adult Value: 90 fL Higher ranges are found in newborns and infants Panels: Hematology MCHC (Mean Corpuscular Hemoglobin Concentration) Hemoglobin x 100 Hematocrit This test measures the average concentration of hemoglobin in red blood cells. It is most valuable in evaluating therapy for anemia because Hemoglobin and Hematocrit are used, not R.B.C. in the calculation. Low MCHC means that a unit of packed R.B.C.s contain less hemoglobin than normal and a high MCHC means that there is more hemoglobin in a unit of R.B.C.s. Increased MCHC is seen in spherocytosis, and not seen in pernicious anemia whereas decreased levels may indicate iron deficiency, blood loss, B6 deficiency, or thalassemia. Normal Adult Range: 32 - 36 % Optimal Adult Value: 34 % Higher ranges are found in newborns and infants Panel: Hematology R.B.C. (Red Blood Cell Count) Red blood cells main function is to carry oxygen to the tissues and to transfer carbon dioxide to the lungs. This process is possible through the R.B.C. containing hemoglobin which combines easily with oxygen and carbon dioxide. Normal Adult Female Range: 3.9 - 5.2 mill/uL Optimal Adult Female Value: 4.55 mill/uL Normal Adult Male Range: 4.2 - 5.6 mill/uL Optimal Adult Male Value: 4.9 mill/uL Lower ranges are found in children, newborns, and infants Panel: Hematology W.B.C. (White Blood Cell Count) White blood cells main function is to fight infection, defend the body by phagocytosis against invasion by foreign organisms, and to produce, or at least transport and distribute, antibodies in the immune response. There are a number of types of leukocytes (see differential) that are classified as follows:

Granulocytes Banded Neutrophils Neutrophils Eosinophils Basophils

Nongranulocyt es Lymphocytes Monocytes

Each cell, or leukocyte, has a different job in the body which is explained in the Differential section. Normal Adult Range: 3.8 - 10.8 thous/uL Optimal Adult Value: 7.3 thous/uL Higher ranges are found in children, newborns, and infants. Panels: Allergy, Hematology PLATELET COUNT Platelets (also known as thrombocytes) are the smallest formed elements of the blood. They are vital to coagulation of the blood to prevent excessive bleeding. Elevated levels suggest dehydration or stimulation of the bone marrow where the cells are produced and decreased levels may indicate an immune system failure, drug reactions, B12, or folic acid deficiency. Normal Adult Range: 130 - 400 thous/uL Optimal Adult Value: 265 thous/uL Higher ranges are found in children, newborns and infants. Panel: Hematology Electrolytes SODIUM Sodium is the most abundant cation in the blood and its chief base. It functions in the body to maintain osmotic pressure, acid-base balance, and to transmit nerve impulses.

Normal Adult Range: 135 - 146 mmol/L Optimal Adult Value: 140.5 mmol/L Panels: Electrolyte POTASSIUM Potassium is the major intracellular cation in the blood. It, along with sodium, helps to maintain osmotic balance and is also involved in acid-base balance. It is needed for proper nerve and muscle action. Normal Range: 3.5 - 5.5 mmol/L Optimal Adult Value: 4.5 mmol/L Panels: Electrolyte CHLORIDE Chloride's significance relates to its maintenance of cellular integrity through its influence on osmotic pressure, it also helps monitor acid-base balance and water balance. Elevated levels are related to acidosis as well as too much water crossing the cell membrane. Decreased levels with decreased serum albumin may indicate water deficiency crossing the cell membrane (edema). Panels: Electrolyte Normal Adult Range: 95 - 109 mmol/L Optimal Adult Value: 103 mmol/L CO2 (Carbon Dioxide) The CO2 level is related to the respiratory exchange of carbon dioxide in the lungs and is part of the body's buffering system. Generally when used with the other electrolytes, it is a good indicator of acidity and alkalinity. Normal Adult Range: 22 - 32 mmol/L Optimal Adult Value: 27 mmol/L Normal Child Range: 20 - 28 mmol/L Optimal Child Value: 24 mmol/L Panels: Electrolyte

CALCIUM The most abundant mineral in the body, it is involved in bone metabolism, protein absorption, fat transfer muscular contraction, transmission of nerve impulses, blood clotting, and cardiac function. It is highly sensitive to elements such as magnesium, iron, and phosphorus as well as hormonal activity, vitamin D levels, alkalinity and acidity, and many drugs. Normal Adult Range: 8.5 - 10.3 mg/dL Optimal Adult Value: 9.4 mg/dL Panels: Electrolyte, Kidney Function PHOSPHORUS Phosphorus is an abundant element found in most tissues and cells. It is closely related to the calcium level with an inverse relationship. When calcium is increased, phosphorus tends to decrease and vice versa. Careful following of blood draw procedures are necessary because improper handling may cause falsely elevated values. Phosphorus is needed for its buffering action, calcium transport, and osmotic pressure. Normal Adult Range: 2.5 - 4.5 mg/dL Optimal Adult Value: 3.5 mg/dL Normal Child Range: 3 - 6 mg/dL Optimal Child Range: 4.5 mg/dL Panels: Electrolyte, Kidney Function Liver Enzymes sGOT (serum Glutamic-Oxaloacetic Transaminase - AST) Serum Glutamic Oxaloacetic Transaminase or AST (Aspartate Aminotransferase) is an enzyme found primarily in the liver, heart, kidney, pancreas, and muscles. Seen in tissue damage, especially heart and liver, this enzyme is normally elevated. Vitamin B deficiency and pregnancy are two instances where the enzyme may be decreased. Normal Adult Range: 0 - 42 IU/L Optimal Adult Value: 21 IU/L Panels: Cardiac Marker, Liver Function

sGPT (serum Glutamic-Pyruvic Transaminase - ALT) Serum Glutamic Pyruvic Transaminase or ALT (Alanine Aminotransferase) is an enzyme found primarily in the liver but also to a lesser degree, in the heart and other tissues. It is useful in diagnosing liver function more so than sGOT levels. Decreased sGPT in combination with increased cholesterol levels is seen in cases of a congested liver. Increased levels are also seen in mononucleosis, alcoholism, liver damage, kidney infection, chemical pollutants, or myocardial infarction. Normal Adult Range: 0 - 48 IU/L Optimal Adult Value: 24 IU/L Panels: Liver Function ALKALINE PHOSPHATASE Produced in the cells of the bone and liver with some activity in the kidney, intestine, and placenta. Used extensively as a tumor marker it is also present in bone injury, pregnancy, or skeletal growth (elevated values). Growing children have normally higher levels of this enzyme also. Low levels are sometimes found in hypoadrenia, protein deficiency, malnutrition, and a number of vitamin deficiencies. Normal Adult Range: 20 - 125 IU/L Optimal Adult Value: 72.5 IU/L Normal Child Range: 40 - 400 IU/L Optimal Child Value: 220 IU/L Panels: Liver Function GGT (Gamma-Glutamyltransferase or Gamma-Glutamyl Transpeptidase) Believed to be involved in the transport of amino acids and peptides into cells as well as glutathione metabolism, Gamma-Glutamyl Transferase is mainly found in liver cells and as such is extremely sensitive to alcohol use. Elevated levels may be found in liver disease, alcoholism, bile-duct obstruction, cholangitis, drug abuse, and in some cases excessive magnesium ingestion. Decreased levels can be found in hypothyroidism, hypothalamic malfunction, and low levels of magnesium. Normal Adult Female Range: 0 - 45 IU/L Optimal Female Value: 22.5 IU/L Normal Adult Male Range: 0 - 65 IU/L Optimal Male Value: 32.5 IU/L

Panels: Liver Function LD (Lactic Dehydrogenase - LDH) Lactic dehydrogenase is an intracellular enzyme from particularly in the kidney, heart, skeletal muscle, brain, liver, and lungs. Increases are usually found in cellular death and/or leakage from the cell or, in some cases, it can be useful in confirming myocardial or pulmonary infarction (only in relation to other tests). Decreased levels of the enzyme may be seen in cases of malnutrition, hypoglycemia, adrenal exhaustion, or low tissue or organ activity. Normal Adult Range: 0 - 250 IU/L Optimal Adult Value: 125 IU/L Panels: Cardiac Marker, Kidney Function, Liver Function BILIRUBIN, TOTAL A byproduct of the breakdown of hemoglobin from red blood cells in the liver, bilirubin is a good indication of the liver's function. Excreted into the bile, bilirubin gives the bile its pigmentation. Elevated in liver disease, mononucleosis, hemolytic anemia, low levels of exposure to the sun, and toxic effects to some drugs, decreased levels are seen in people with an inefficient liver, excessive fat digestion, and possibly a diet low in nitrogen bearing foods. Normal Adult Range: 0 - 1.3 mg/dL Optimal Adult Value: 0.65 mg/dL Panels: Liver Function Nitrogen Elements B.U.N. (Blood Urea Nitrogen) The nitrogen component of urea, B.U.N. is the end product of protein metabolism and its concentration is influenced by the rate of excretion. Increases can be caused by excessive protein intake, kidney damage, certain drugs, low fluid intake, intestinal bleeding, exercise, or heart failure. Decreased levels may be due to a poor diet, malabsorption, liver damage, or low nitrogen intake. Normal Adult Range: 7 - 25 mg/dL Optimal Adult Value: 16 mg/dL Panels: Kidney Function, Nitrogen

CREATININE Creatinine is the waste product of muscle metabolism. Its level is a reflection of the body's muscle mass. Low levels are sometimes seen in kidney damage, protein starvation, liver disease, or pregnancy. Elevated levels are sometimes seen in kidney disease due to the kidneys job of excreting creatinine, muscle degeneration, and some drugs involved in impairment of kidney function. Normal Adult Range: 0.7 - 1.4 mg/dL Optimal Adult Value: 1.05 mg/dL Panels: Kidney Function, Nitrogen URIC ACID Uric acid is the end product of purine metabolism and is normally excreted through the urine. High levels are noted in gout, infections, kidney disease, alcoholism, high protein diets, and with toxemia in pregnancy. Low levels may be indicative of malabsorption, a diet low in purines, liver damage, or an overly acid kidney. Normal Adult Female Range: 2.5 - 7.5 mg/dL Optimal Adult Female Value: 5.0 mg/dL Normal Adult Male Range: 3.5 - 7.5 mg/dL Optimal Adult Male Value: 5.5 mg/dL Panels: Kidney Function, Nitrogen Protein PROTEIN, TOTAL Proteins are the most abundant compound in serum. The protein makeup of the individual is of important diagnostic significance because of protein's involvement in enzymes, hormones, and antibodies as well as osmotic pressure balance, maintaining acid-base balance, and as a reserve source of nutrition for the body's tissues and muscles. The major serum proteins measured are Albumin and Globulin (alpha1, alpha2, beta, and gamma). Decreased levels may be due to poor nutrition, liver disease, malabsorption, diarrhea, or severe burns. Increased levels are seen in lupus, liver disease, chronic infections, alcoholism, leukemia, tuberculosis amongst many others. Careful review of the individuals albumin, globulin, and A/G ratio are recommended. Normal Adult Range: 6.0 - 8.5 g/dL Optimal Adult Value: 7.25 g/dL

Panels: Kidney Function, Liver Function, Protein ALBUMIN Albumin is the major constituent of serum protein (usually over 50%). It is manufactured by the liver from the amino acids taken from the diet. It helps in osmotic pressure regulation, nutrient transport, and waste removal. High levels are rarely seen and are primarily due to dehydration. Low levels are seen in poor diets, diarrhea, fever, infection, liver disease, inadequate iron intake, third-degree burns and edemas, and hypocalcemia. Panels: Kidney Function, Liver Function, Protein Normal Adult Range: 3.2 - 5.0 g/dL Optimal Adult Value: 4.1 g/dL GLOBULIN Globulin, a larger protein than albumin, is important for its immunologic responses, especially its gamma component (IgA, IgG, IgM, and IgE). Globulins have many diverse functions such as, the carrier of some hormones, lipids, metals, and antibodies. When chronic infections, liver disease, rheumatoid arthritis, myelomas, and lupus are present, elevated levels are seen. Lower levels may be found in immune compromised patients, poor dietary habits, malabsorption, and liver or kidney disease. Normal Adult Range: 2.2 - 4.2 g/dL (calculated) Optimal Adult Value: 3.2 g/dL Panels: Allergy, Kidney Function, Liver Function, Protein A/G RATIO (Albumin/Globulin Ratio) A/G ratio is an important indicator of disease states although a high level is not considered clinically significant. Normal Adult Range: 1.1 - 2.4 (calculated) Optimal Adult Value: 1.9 Panels: Protein, Kidney Function, Liver Function, Ratios Lipids

CHOLESTEROL Cholesterol is a critical fat that is a structural component of cell membrane and plasma lipoproteins, and is important in the synthesis of steroid hormones, glucocorticoids, and bile acids. Mostly synthesized in the liver, some is absorbed through the diet, especially one high in saturated fats. High density lipoproteins (HDL) is desired as opposed to the low density lipoproteins (LDL), two types of cholesterol. Elevated cholesterol has been seen in artherosclerosis, diabetes, hypothyroidism, and pregnancy. Low levels are seen in depression, malnutrition, liver insufficiency, malignancies, anemia, and infection. Normal Adult Range: 120 - 240 mg/dL Optimal Adult Value: 180 mg/dL Panels: Cardiac Marker, Kidney Function, Lipids, Liver Function TRIGLYCERIDES Triglycerides, stored in adipose tissues as glycerol, fatty acids and monoglycerides, are reconverted as triglycerides by the liver. Ninety percent of the dietary intake and 95% of the fat stored in tissues are triglycerides. Increased levels may be present in artherosclerosis, hypothyroidism, liver disease, pancreatitis, myocardial infarction, metabolic disorders, toxemia, and nephrotic syndrome. Decreased levels may be present in chronic obstructive pulmonary disease, brain infarction, hyperthyroidism, malnutrition, and malabsorption. Normal Adult Range: 0 - 200 mg/dL Optimal Adult Value: 100 mg/dL Panels: Cardiac Marker, Lipids LDL (Low Density Lipoprotein) LDL is the cholesterol rich remnants of the lipid transport vehicle VLDL (very-low density lipoproteins) there have been many studies to correlate the association between high levels of LDL and arterial artherosclerosis. Due to the expense of direct measurement of LDL a calculation, known as the Friedewald formula is used. It is Total Cholesterol - HDL Cholesterol - (Triglycerides/5). When triglyceride levels are greater than 400 mg/dL, this calculation is not accurate. Normal Adult Range: 62 - 130 mg/dL Optimal Adult Value: 81 mg/dL Panels: Cardiac Marker, Lipids

HDL (High Density Lipoprotein) HDL or High-density lipoprotein is the cholesterol carried by the alpha lipoproteins. A high level of HDL is an indication of a healthy metabolic system if there is no sign of liver disease or intoxication. The two mechanisms that explain how HDL offers protection against chronic heart disease are that 1. HDL inhibits cellular uptake of LDL and 2. serves as a carrier that removes cholesterol from the peripheral tissues and transports it back to the liver for catabolism and excretion. Normal Adult Range: 35 - 135 mg/dL Optimal Adult Value: >85 mg/dL Panels: Cardiac Marker, Lipids CHOLESTEROL/LDL RATIO The ratio of total cholesterol and LDL (low density lipoprotein). Normal Adult Range: 1 - 6 Optimal Adult Value: 3.0 Panels: Cardiac Marker, Ratios Ratios ANION GAP (Sodium + Potassium - CO2 - Chloride) The anion gap is used to measure the concentration of cations (sodium and potassium) and the anions (chloride and CO2) in the extracellular fluid of the blood. There are numerous clinical implications that can be gathered from the Anion Gap. An increased measurement is associated with metabolic acidosis due to the overproduction of acids (a state of alkalinity is in effect). Decreased levels may indicate metabolic alkalosis due to the overproduction of alkaloids (a state of acidosis is in effect). Normal Adult Range: 4 - 14 (calculated) Optimal Adult Value: 9 Panels: Ratios B.U.N./CREATININE A high value in this calculation is normally indicative of too much B.U.N. being formed and a low value may show that the B.U.N. is low or that the creatinine is

not being cleared effectively by the kidney. This calculation is a good measurement of kidney and liver function. Normal Adult Range: 6 - 25 (calculated) Optimal Adult Value: 15.5 Panels: Nitrogen, Ratios CALCIUM/PHOSPHORUS Due to the delicate balance between calcium and phosphorus in the system, this calculation is helpful in noting subtle and acute imbalances in the relationship between the two elements. Normal Adult Range: 2.3 - 3.3 (calculated) Optimal Adult Value: 2.8 Normal Child Range: 1.3 - 3.3 (calculated) Optimal Child Value: 2.3 Panels: Ratios SODIUM/POTASSIUM As the two major blood electrolytes, sodium as the extracellular cation and potassium as the intracellular cation, this is an important ratio to review and act upon when subtle or acute imbalances are noted. Normal Adult Range: 26 - 38 (calculated) Optimal Adult Value: 32 Panels: Ratios Differential NEUTROPHILS and NEUTROPHIL COUNT Also known as Granulocytes or poly-segmented neutrophils, this is the main defender of the body against infection and antigens. High levels may indicate an active infection, a low count may indicate a compromised immune system or depressed bone marrow (low neutrophil production. Normal Adult Range: 48 - 73 % Optimal Adult Value: 60.5 %

Normal Child Range: 30 - 60 % Optimal Child Value: 45 % Panels: Differential, Differential Count LYMPHOCYTES and LYMPHOCYTE COUNT Lymphocytes are involved in protection of the body from viral infections such as measles, rubella, chickenpox, or infectious mononucleosis. Elevated levels may indicate an active viral infection and a depressed level may indicate an exhausted immune system or, if the neutrophils are elevated, an active infection. Normal Adult Range: 18 - 48 % Optimal Adult Value: 33 % Normal Child Range: 25 - 50 % Optimal Child Value: 37.5 % Panels: Allergy, Differential, Differential Count MONOCYTES and MONOCYTE COUNT These cells are helpful in fighting severe infections and are considered the body's second line of defense against infection and are the largest cells in the blood stream. Elevated levels are seen in tissue breakdown, chronic infections, carcinomas, leukemia (monocytic), or lymphomas. Low levels are indicative of a good state of health. Normal Adult Range: 0 - 9 % Optimal Adult Value: 4.5 % Panels: Differential, Differential Count, Allergy EOSINOPHILS and EOSINOPHIL COUNT Eosinophils are used by the body to protect against allergic reactions and parasites. Therefore, elevated levels may indicate an allergic response. A low count is normal. Normal Adult Range: 0 - 5 % Optimal Adult Value: 2.5 % Panels: Allergy, Differential, Differential Count

BASOPHILS and BASOPHIL COUNT Basophilic activity is not fully understood but it is known to carry histamine, heparin, and serotonin. High levels are found in allergic reactions, low levels are normal. Normal Adult Range: 0 - 2 % Optimal Adult Value: 1 % Panels: Differential, Differential Count Thyroid THYROXINE (T4) Thyroxine is the thyroid hormone that contains four atoms of iodine. It is used to evaluate thyroid function. It is the direct measurement of total T4 concentration in the blood serum. Increased levels are found in hyperthyroidism, acute thyroiditis, and hepatitis. Low levels can be found in Cretinism, hypothyroidism, cirrhosis, malnutrition, and chronic thyroiditis. Normal Adult Range: 4 - 12 ug/dL Optimal Adult Value: 8 ug/dL Panels: Thyroid T-UPTAKE (T3-Uptake) This test is an indirect measurement of unsaturated thyroxine binding globulin in the blood. Increased levels are found in hyperthyroidism, severe liver disease, metastatic malignancy, and pulmonary insufficiency. Decreased levels are found in hypothyroidism, normal pregnancy, and hyperestrogenis status. Normal Adult Range: 27 - 47% Optimal Adult Value: 37 % Panels: Thyroid FREE T4 INDEX (T7) This index is a calculation used to correct the estimated total thyroxine for the amount of thyroxine binding globulin present. It uses the T4 value and the Tuptake ratio.

Normal Adult Range: 4 - 12 Optimal Adult Value: 8 Panels: Thyroid THYROID-STIMULATING HORMONE (TSH) TSH, produced by the anterior pituitary gland, causes the release and distribution of stored thyroid hormones. When T4 and T3 are too high, TSH secretion decreases, when T4 and T3 are low, TSH secretion increases. Normal Adult Range: 0.5 - 6 mIU/L Optimal Adult Value: 1.75 mIU/L Panels: Thyroid Other GLUCOSE (Fasting) Glucose, formed by the digestion of carbohydrates and the conversion of glycogen by the liver, is the primary source of energy for most cells. It is regulated by insulin, glucagon, thyroid hormone, liver enzymes, and adrenal hormones. It is elevated in diabetes, liver disease, obesity, pancreatitis, due to steroid medications, or during stress. Low levels may be indicative of liver disease, overproduction of insulin, hypothyroidism, or alcoholism. Normal Adult Range: 60 - 109 mg/dL Optimal Adult Value: 87.5 mg/dL IRON, TOTAL Iron is necessary for the formation of some proteins, hemoglobin, myoglobin, and cytochrome. Also, it is necessary for oxygen transport, cellular respiration, and peroxide deactivation. Low levels are seen in many anemias, copper deficiencies, low vitamin C intake, liver disease, chronic infections, high calcium intake, and women with heavy menstrual flows. High levels are seen in hemochromatosis, liver damage, pernicious anemia, and hemolytic anemia. Normal Adult Range: 30 - 170 ug/dL Optimal Adult Value: 100 ug/dL

Blood type A blood type is a description an individual's characteristics of red blood cells due to substances (carbohydrates and proteins) on the cell membrane. The two most important classifications to describe blood types in humans are ABO and Rh factor. There are 46 other known antigens, most of which are much rarer than ABO and Rh. Blood transfusions from incompatible groups can cause an immunological transfusion reaction , resulting in hemolytic anemia, renal failure, shock, and death. ABO Humans have the following blood types along with their respective antigens and antibodies:

Individuals with type A blood have red blood cells with antigen A on their surface and produce antibodies against antigen B in their blood serum. Using the blood compatibility chart below, for example, an A-negative person cannot receive blood except from another A-negative person or from an O-negative person. Individuals with type B blood have the opposite arrangement, antigen B on the cell and produce antibodies to substance A in their serum. Type AB people have red blood cells with both antigens A and B, and do not produce antibodies against either substance in their serum. Therefore, a person with type AB blood can safely receive any ABO type blood and is called a "universal receiver", but cannot donate blood except to the corresponding AB type people shown in the blood compatibility table below. Type O people have red blood cells with neither antigen, but produce antibodies against both types of antigens. Because of this arrangement, type O can be safely given to any person with any ABO blood type. Hence, a person with type O blood is said to be a "universal donor" but cannot receive blood except from the corresponding O type people shown in the blood compatibility table below. Thus, for example, an Onegative person cannot receive blood except from another O-negative person.

Overall, the O blood type is the most common blood type in the world, although in some areas, such as Norway, the A group dominates. The A antigen is overall more common than the B antigen. Since the AB blood type requires the presence of both

A and B antigens, the AB blood type is the rarest of the ABO blood types. There are known racial and geographic distributions of the ABO blood types [1]. The precise reason why people are born with antibodies against an antigen they have never been exposed to is unknown. It is believed that some bacterial antigens are similar enough to the A and B glycoproteins, and that antibodies created against the bacteria will react to ABO-incompatible blood cells. Apart from on red blood cells, the ABO antigen is also expressed on the glycoprotein von Willebrand factor (vWF), which participates in hemostasis (control of bleeding). In fact, blood type O predisposes very slightly to bleeding, as vWF is degraded more rapidly. Austrian scientist Karl Landsteiner was awarded the Nobel Prize in Physiology or Medicine in 1930 for his work in discovering ABO blood types. Rhesus Another characteristic of blood is Rhesus factor or Rh factor. It is named after the Rhesus Monkey, where the factor was first identified in 1940. Someone either has or does not have the Rh factor on the surface of their red blood cells. This is indicated as + or -. This is often combined with the ABO type. Type O+ blood is most common, though in some areas type A prevails, and there are other areas in which as many as 80 percent of the people are type B. Matching the Rhesus factor in the ABO system is very important, as mismatching (an Rh positive donor to an Rh negative recipient) will cause hemolysis. The converse is not true: Rh+ patients do not react to Rh- blood. Rh disease occurs when an Rh negative mother who has already had an Rh positive child (or an accidental Rh+ blood transfusion) carries another Rh positive child. After the first pregnancy, the mother develops antibodies against Rh+ red blood cells, which cross the placenta and hemolyses the blood of the second child. This reaction doesn't always occur and is less likely to occur if the child carries either the A or B antigen and the mother does not. In the past, Rh incompatibility could result in stillbirth or death of the mother. Rh incompatibility was until recently the most common cause of long term disability in the United States. At first, this was treated by transfusing the blood of infants who survived. At present, it can be treated with certain anti-Rh(+) antisera, the most common of which is Rhogam (anti-D). It can be anticipated by determining the blood type of every child of a RhD- mother; if it is Rh+, the mother is treated with anti-D to prevent development of antibodies against Rh+ red blood cells. Inheritance

ABO Blood groups are inherited from both parents. The ABO blood type is controlled by a single gene with three alleles: i, A, and B. The gene encodes a glycosyltransferase , an enzyme that modifies the carbohydrate content of the red blood cell antigens. The gene is located on the long arm of the ninth chromosome (9q34). A allele gives type A, B gives type B, and i gives type O. A and B are dominant over i, so ii people have type O, AA or Ai have A, BB or Bi have type B. AB people have both phenotypes because A and B express a special dominance relationship: codominance. Thus, it is usually impossible for a type AB parent to have a type O child (it is, however, no direct proof of illegitimacy). Evolutionary biologists theorize that the A allele evolved earliest, followed by O and then B. This chronology accounts for the percentage of people worldwide with each blood type. It is consistent with the accepted patterns of early population movements and varying prevalent blood types in different parts of the world. (For instance, B is very common in populations of Asian descent, but rare in ones of European descent.) Rhesus Rh (or the D antigen) is inherited on one locus (on the short arm of the first chromosome, 1p36.2-p34) with two alleles, of which Rh+ is dominant and Rhrecessive. The gene codes for a polypeptide on the red cell membrane. Rhindividuals (dd genotype) do not produce this antigen, and may be sensitized to Rh+ blood. Two very similar epitopes, Cc and Ee, appear to be closely related to Rh. Rare phenotypes Bombay phenotype The rare individuals with Bombay phenotype do not express substance H on their red blood cells and therefore do not bind A or B antigens. Instead, they produce antibodies to H substance (which is present on all red cells except those of hh phenotype) as well as to both A and B antigens and are therefore compatible only with other hh donors. Individuals with Bombay phenotype blood groups can only be transfused with blood from other Bombay phenotype individuals. Given that this condition is very rare to begin with, any person with this blood group, who needs an urgent blood

transfusion, may be simply out of luck, as it would be quite unlikely that any blood bank would have any in stock. Patients who test as type O may have the Bombay phenotype: they have inherited two recessive alleles of the H gene, (their blood group is Oh and their genotype is "hh"), and so do not produce the "H" protein that is the precursor to the "A" and "B" antigens. It then no longer matters whether the A or B enzymes are present or not, as no A or B antigen can be produced since the precursor antigen is not present. Despite the designation O, Oh -ve is not a sub-group of any other group, not even O -ve or O +ve. When this Blood group was first encountered, it was found not to be of either group A or B and so was thought to be of Group O. But on further test, it did not match even for O-ve or O+ve because of the absence of Antigen 'h'. The H antigen is a precursor to the A and B antigens. For instance, the B allele must be present to produce the B enzyme that modifies the H antigen to become the B antigen. It is the same for the A allele. However, if only recessive alleles for the H antigen are inherited (hh), as in the case above, the H antigen will not be produced. Subsequently, the A and B antigens also will not be produced. The result is an O phenotype by default since a lack of A and B antigens is the O type. The name "Bombay group" originates from the city of Bombay, now known as Mumbai, in India. The blood phenotype was first discovered in Bombay. Compatibility Blood donors and blood recipients must have compatible blood types. O- is the universally compatible blood type. The chart below illustrates how people with different blood types can receive or donate other blood (X means compatible). An A- person, for example, can receive either O- or A-, and can donate to people with AB+, AB-, A+ or A- blood.

In general, people with type O Rh- are referred to as universal donors, as their blood can be Blood compatibility chart transfused to anyone in need. It Donor is thus the most highly sought Recipie after blood type O B B A AB AB nt OAin blood banks + - + + + and hospitals. A type AB Rh+ is AB+ X X X X X X X X referred to as a universal ABX X X X receiver because he or she can A+ X X X X receive blood of any type. AX X B+ Frequency Blood types are BX X X X not evenly distributed throughout the human population. O+ is the most common, AB- is the rarest. There are also variations in blood-type distribution within human subpopulations. X X X X X

O+ Typ Frequenc e yOO+ A+ B+ OA38% 34% 9% 7% 6% 3% 2% 1%

Other blood types Other blood type systems exist to describe the presence or absence of other antigens. Many are named after the patients in whom they were initially encountered.

AB+ BAB-

Diego positive blood is found only among East Asians and Native Americans.

MNS systems gives blood types of M, N, and MN. It has use in tests of maternity or paternity. Duffy negative blood gives partial immunity to malaria. The Lutheran system describes a set of 21 antigens. Other systems include Colton, Kell, Kidd, Lewis, LandsteinerWiener, P, Yt or Cartwright, XG, Scianna, Dombrock, Chido/Rodgers, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, and JMH.

Social significance In Japan (and to a certain extent in Taiwan) a popular belief is that personality is related to blood type. Visitors to the country are often surprised to be asked their blood type by people they encounter. Japanese people are also often surprised when foreigners say that they do not know their own blood type. Whilst many Japanese people understand that blood typing doesn't hold any social significance in Western societies, others may conclude that the visitor is too "ashamed" of their blood type to reveal it to others. Furthermore, from the preponderance of some blood types in a population, Japanese "experts" claim to be able to deduce the character of that population. The experts also believe that they can calculate how well the blood types of different people match, a Japanese employer could therefore aim to get a proper mix of blood types among their personnel. Some nationalisms such as the Basque one have used the different proportion of blood types in different regions or populations as a mark of different race. In the United States, few African Americans donate blood, resulting in a shortage of U-negative and Duffy-negative blood for African American patients. References

Landsteiner K. Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe. Zentralblatt Bakteriologie 1900;27:357-62.

Metal Complexes in the Blood for Oxygen Transport : Introduction to the Chemistry and Physiology of Blood Our bodies consist of cells that are organized into many specialized organs and tissues to perform a variety of functions. Our stomachs digest food so that the nutrients contained in the food can be distributed to the rest of the body. Our lungs take in the oxygen needed by the body from the air and release carbon dioxide as a waste product. Our muscles allow the body to move. Our brains coordinate all of these (and many other) activities of the body. These processes are based upon

many different chemical reactions, and the sum total of the chemical reactions in the body is known as the body's metabolism. The metabolism includes the reactions needed for normal everyday activities such as eating, sleeping, and studying. When we exercise, the metabolism increases to allow our body to cope with the increased demands and stress of exercising. All of our specialized body parts are united by their fundamental need for a particular chemical environment that will enable the body's metabolic reactions. This environment must include a supply of nutrients (e.g., sugars and vitamins, to supply the building blocks for cells and enable biochemical reactions) and oxygen (to provide energy for the body; the process of using oxygen to make the body's energy supply is described in the Chem 152 tutorial, "Energy for the Body: Oxidative Phosphorylation"), and the ability to eliminate the waste products of the body's metabolic activities. This environment is provided by bathing our body's cells in blood. Blood is part of the body's circulatory system, and thus is continually being pumped through our bodies as long as we are alive. The blood distributes oxygen and nutrients to the many different cells in the body, carries CO2 generated by the cells to the lungs for exhalation, and carries other waste products to the kidneys and liver for processing and elimination. Many finely tuned chemical processes occur in the blood to allow the blood to carry out all of these functions and provide for the needs of the body. The tutorials in Chem 151 and 152 will describe several of these vital chemical processes, such as the release of iron in controlled amounts to the blood ("Iron Use and Storage in the Body: Ferritin and Molecular Representations" tutorial), removal of waste products from the blood ("Maintaining the Body's Chemistry: Dialysis in the Kidneys" tutorial), and the regulation of the levels of CO2 and H+ to control the pH of the blood ("Blood, Sweat, and Buffers: pH Regulation During Exercise" tutorial). In this tutorial, we will study one of the most important functions of blood, the transport of oxygen from the lungs to the other cells of the body (e.g., muscle cells) that perform metabolic functions. Oxygen Transport via Metal Complexes An adult at rest consumes the equivalent of 250 ml of pure oxygen per minute. This oxygen is used to provide energy for all the tissues and organs of the body, even when the body is at rest. The body's oxygen needs increase dramatically during exercise or other strenuous activities. The oxygen is carried in the blood from the lungs to the tissues where it is consumed. However, only about 1.5% of the oxygen transported in the blood is dissolved directly in the blood plasma. Transporting the large amount of oxygen required by the body, and allowing it to leave the blood when it reaches the tissues that demand the most oxygen, require a more sophisticated mechanism than simply dissolving the gas in the blood. To meet this challenge, the body is equipped with a finely-tuned transport system that centers on the metal complex heme.

Metal Complexes in the Body The ability of metal ions to coordinate with (bind) and then release ligands in some processes, and to oxidize and reduce in other processes makes them ideal for use in biological systems. The most common metal used in the body is iron, and it plays a central role in almost all living cells. For example, iron complexes are used in the transport of oxygen in the blood and tissues. Metal-ion complexes consist of a metal ion that is bonded via "coordinate-covalent bonds" (Figure 1) to a small number of anions or neutral molecules called ligands. For example the ammonia (NH3) ligand used in this experiment is a monodentate ligand; i.e., each monodentate ligand in a metal-ion complex possesses a single electron-pair-donor atom and occupies only one site in the coordination sphere of a metal ion. Some ligands have two or more electron-pair-donor atoms that can simultaneously coordinate to a metal ion and occupy two or more coordination sites; these ligands are called polydentate ligands. They are also known as chelating agents (from the Greek word meaning "claw"), because they appear to grasp the metal ion between two or more electron-pair-donor atoms. The coordination number for a metal refers to the total number of occupied coordination sites around the central metal ion (i.e., the total number of metalligand bonds in the complex).

Figure 1 You have already learned that a covalent bond forms when electrons are shared between atoms. A coordinate-covalent bond (represented by a green arrow in this diagram) forms when both of the shared electrons come from the same atom, called the donor atom (blue). An anion or molecule containing the donor atom is known as a ligand. The top illustration shows a coordinate-covalent bond between a metal ion (e.g., Fe, shown in red) and a monodentate ligand (a ligand that contains only one electron-pair-donor atom, shown in light blue). The bottom illustration shows a metal ion with coordinate-covalent bonds to a bidentate ligand (a ligand that contains two donor atoms simultaneously coordinated to the metal ion, shown in yellow). Oxygen-Carrying Protein in the Blood: Hemoglobin Hemoglobin is the protein that transports oxygen (O2) in human blood from the lungs to the tissues of the body. Proteins are formed by the linking of amino acids into polypeptide chains. An individual amino acid in a protein is known as a "residue." The arrangement and interactions of the amino-acid residues within the protein determine the protein's shape and contribute substantially to its function. Hemoglobin is a globular protein (i.e., folded into a compact, nearly spherical shape) and consists of four subunits, as shown in Figure 2. Each protein subunit is an individual molecule that joins to its neighboring subunits through intermolecular interactions. (These subunits are also known as peptide chains. You will learn more about the nature of amino acids and peptide subunits in the tutorial entitled, "Iron Use and Storage in the Body: Ferritin and Molecular Representations".)

Figure 2 This is a molecular model of hemoglobin with the subunits displayed in the ribbon representation. A ribbon representation traces the backbone atoms of a protein and is often used to represent its three-dimensional structure. The four heme groups are displayed in the ball-and-stick representation. Note: The coordinates for the hemoglobin protein (in this and subsequent molecular representations of all or part of the protein) were determined using x-ray crystallography, and the image was rendered using SwissPDB Viewer and POV-Ray (see References). Note: To view the molecule interactively, please use Chime, and click on the button to the left. Chime currently works in IE 6.0, Netscape 4.75 or Netscape 4.79. It does not work any other version of Netscape. You will need to check the MDL Website periodically for
any updates.

To understand the oxygen-binding properties of hemoglobin, we will focus briefly on the structure of the protein and the metal complexes embedded in it. The Protein Subunit Each subunit in Figure 2 contains regions with a coiled shape; many of the amino acids that make up the polypeptide chain interact to form this particular structure, called an alpha helix. In an alpha helix (Figure 3), each amino acid is "hydrogenbonded" to the amino acid that is four residues ahead of it in the chain. In hemoglobin, the hydrogen-bonding interaction occurs between the H of an -NH group and the O of a -CO group of the polypeptide backbone chain; the amino-acid side chains extend outward from the backbone of the helix. Approximately 75% of the amino-acid composition of hemoglobin adopts an alpha-helical structure. Another common structural motif is the beta-pleated sheet, in which amino acids line up in straight parallel rows.

Figure 3 This is a molecular model of the alpha-helix structure in a subunit of hemoglobin. The blue strands are a ribbon representation to emphasize the helical structure. The green dotted lines show the hydrogen bonding between the -NH and -CO functional groups.

Note: To view the molecule interactively, please use Chime, and click on the button above. Chime currently works in IE 6.0, Netscape 4.75 or Netscape 4.79. It does not work any other version of Netscape. You will need to check the MDL Website periodically for any updates.

Please click on the pink button above to view a QuickTime movie showing a rotation of the alpha-helix structure shown in Figure 3. Click the blue button below to download QuickTime 4.0 to view the movie.

The Heme Group In hemoglobin, each subunit contains a heme group, which is displayed using the ball-and-stick representation in Figure 2. Each heme group contains an iron atom that is able to bind to one oxygen (O2) molecule. Therefore, each hemoglobin protein can bind four oxygen molecules. One of the most important classes of chelating agents in nature are the porphyrins. A porphyrin molecule can coordinate to a metal using the four nitrogen atoms as electron-pair donors, and hence is a polydentate ligand (see Figure 1). Heme is a porphyrin that is coordinated with Fe(II) and is shown in Figure 4.

Figure 4 On the left is a three-dimensional molecular model of heme coordinated to the histidine residue (a monodentate ligand, see Figure 1) of the hemoglobin protein. On the right is a twodimensional drawing of heme coordinated to the histidine residue, which is part of the hemoglobin protein. In this figure, the protein is deoxygenated; i.e., there is no oxygen molecule bound to the heme group. Note: The coordinate-covalent bonds between the central iron atom and the nitrogens from the porphyrin are gold; the coordinatecovalent bond between the central iron atom and the histidine residue is green. In the three-dimensional model, the carbon atoms are are gray, the iron atom is dark red, the nitrogen atoms are dark blue, and the oxygen atoms are light red. The rest of the hemoglobin protein is purple. Note: To view the molecule interactively, please use Chime, and click on the button to the left. Chime currently works in IE 6.0, Netscape 4.75 or Netscape 4.79. It does not work any other version of Netscape. You will need to check the MDL Website periodically for
any updates.

In the body, the iron in the heme is coordinated to the four nitrogen atoms of the porphyrin and also to a nitrogen atom from a histidine residue (one of the amino-

acid residues in hemoglobin) of the hemoglobin protein (see Figure 4). The sixth position (coordination site) around the iron of the heme is occupied by O2 when the hemoglobin protein is oxygenated. Questions on the Oxygen-Carrying Protein in the Blood: Hemoglobin

One peptide subunit in hemoglobin contains 141 amino-acid residues. If the subunit were stretched out, it would measure approximately 49 nm in length. However, the longest dimension of the subunit in hemoglobin is only about 5 nm. Briefly, explain how alpha helices may help account for this difference in length. What is the coordination number of Fe in the oxygenated heme group? Briefly, justify your answer by describing the ligands to which Fe is coordinated.

Conformational Changes Upon Binding of Oxygen Careful examination of Figure 4 shows that the heme group is nonplanar when it is not bound to oxygen; the iron atom is pulled out of the plane of the porphyrin, toward the histidine residue to which it is attached. This nonplanar configuration is characteristic of the deoxygenated heme group, and is commonly referred to as a "domed" shape. The valence electrons in the atoms surrounding iron in the heme group and the valence electrons in the histidine residue form "clouds" of electron density. (Electron density refers to the probability of finding an electron in a region of space.) Because electrons repel one another, the regions occupied by the valence electrons in the heme group and the histidine residue are pushed apart. Hence, the porphyrin adopts the domed (nonplanar) configuration and the Fe is out of the plane of the porphyrin ring (Figure 5, left). However, when the Fe in the heme group binds to an oxygen molecule, the porphyrin ring adopts a planar configuration and hence the Fe lies in the plane of the porphyrin ring (Figure 5, right).

Figure 5 On the left is a schematic diagram showing representations of electron-density clouds of the deoxygenated heme group (pink) and the attached histidine residue (light blue). These regions of electron density push one another apart, and the iron atom in the center is drawn out of the plane. (The nonplanar shape of the heme group is represented by the bent line.) On the right is a schematic diagram showing representations of electron-density clouds of the oxygenated heme group (pink), the attached histidine residue (light blue), and the attached oxygen molecule (gray). The oxygenated heme assumes a planar configuration, and the central iron atom occupies a space in the plane of the heme group (depicted by a straight red line). The shape change in the heme group has important implications for the rest of the hemoglobin protein, as well. When the iron atom moves into the porphyrin plane upon oxygenation, the histidine residue to which the iron atom is attached is drawn closer to the heme group. This movement of the histidine residue then shifts the position of other amino acids that are near the histidine (Figure 6). When the amino acids in a protein are shifted in this manner (by the oxygenation of one of the heme groups in the protein), the structure of the interfaces between the four subunits is altered. Hence, when a single heme group in the hemoglobin protein becomes oxygenated, the whole protein changes its shape. In the new shape, it is easier for the other three heme groups to become oxygenated. Thus, the binding of one molecule of O2 to hemoglobin enhances the ability of hemoglobin to bind more O2 molecules. This property of hemoglobin is known as "cooperative binding."

Figure 6 This figure shows the heme group and a portion of the hemoglobin protein that is directly attached to the heme. When hemoglobin is deoxygenated (left), the heme group adopts a domed configuration. When hemoglobin is oxygenated (right), the heme group adopts a planar configuration. As shown in the figure, the conformational change in the heme group causes the protein to change its conformation, as well. Please click on the pink button below to view a QuickTime movie showing how the amino acid residues near the heme group in hemoglobin shift as the heme group converts between the nonplanar (domed) and the planar conformation by binding and releasing a molecule of O2. Click the blue button below to download QuickTime 4.0 to view the movie.

Questions on Conformational Changes Upon Binding of Oxygen:

Explain, in terms of electron repulsion, why the heme group adopts a nonplanar (domed) configuration upon deoxygenation. Explain how a change in the heme group configuration causes the entire hemoglobin subunit to change shape.

Spectroscopy and the Color of Blood The changes that occur in blood upon oxygenation and deoxygenation are visible not only at the microscopic level, as detailed above, but also at the macroscopic level. Clinicians have long noted that blood in the systemic arteries (traveling from the heart to the oxygen-using cells of the body) is red-colored, while blood in the systemic veins (traveling from the oxygen-using cells back to the heart) is blue-colored (see Figure 7). The blood in the systemic arteries is oxygen-rich; this blood has just traveled from the lungs (where it picked up oxygen inhaled from the air) to the heart, and then is pumped throughout the body to deliver its oxygen to the body's cells. The blood in the systemic veins, on the other hand, is oxygen-poor; it has unloaded its oxygen to the body's cells (exchanging the O2 for CO2, as described below), and must now return to the lungs to replenish the supply of oxygen. Hence, a simple macroscopic observation, i.e., noting the color of the blood, can tell us whether the blood is oxygenated or deoxygenated. What causes this color change in the blood? We know that the shape of the heme group and the hemoglobin protein change, depending on whether hemoglobin is oxygenated or deoxygenated. The two conformations must have different light-absorbing properties. The oxygenated conformation of hemoglobin must absorb light in the blue-green range, and reflect red light, to account for the red appearance of oxygenated blood. The deoxygenated conformation of hemoglobin must absorb light in the orange range, and reflect blue light, to account for the bluish appearance of deoxygenated blood. We could use a spectrophotometer to examine a dilute solution of blood and determine the wavelength of light absorbed by each conformation. For an approximate prediction of the wavelength of light absorbed and for the colors of light absorbed for a given complementary color, a table such as Table 1 in the introduction to the Experiment ("Relations Between Electronic Transition Energy and Color") could be used.

Questions on Spectroscopy and the Color of Blood

Propose an explanation for why the change in heme group conformation results in a color change. A researcher prepares two solutions of deoxygenated hemoglobin. One solution is ten times as concentrated as the other solution. The researcher then obtains absorption spectra for the two solutions. Do you expect the wavelength of maximum absorption ( max) to be the same or different for the two solutions? If max is different for the two solutions, indicate which solution will have a higher max. Briefly, explain your reasoning. Do you expect the absorbance (A) at max to be the same or different for the two solutions? If the absorbance is different for the two solutions, indicate which solution will have a higher absorbance. Briefly, explain your reasoning.

a. b.

The Effect of CO2 and H+ on O2 Binding In 1904, Christian Bohr discovered that increased concentrations of CO2 and H+ promote the release of O2 from hemoglobin in the blood. This phenomenon, known as the Bohr effect, is a highly adaptive feature of the body's blood-gas exchange mechanism. The blood that is pumped from the heart to the body tissues and organs (other than the lungs) is rich in oxygen (Figure 7). These tissues require oxygen for their metabolic activities (e.g., muscle contraction). Hence, it is necessary for oxygen to remain bound to hemoglobin as the blood travels through the arteries (so that it can be carried to the tissues), but be easily removable when the blood passes through the capillaries feeding the body tissues. CO2 and H+ are produced from metabolic activities of the body, and so the concentration of these species is high in the metabolically active tissues of the body. Thus, the tissues that perform the most metabolic activity (and therefore require the largest amount of O2) produce large quantities of CO2 and H+, facilitating the release of O2 from the blood where the O2 is most needed. In the lungs, the reverse effect occurs: high concentrations of O2 cause the release of CO2 from hemoglobin. 1. Blood rich in carbon dioxide is pumped from the heart into the lungs through the pulmonary arteries. (Arteries are blood vessels carrying blood away from the heart; veins are blood vessels carrying blood to the heart.) In the lungs, CO2 in the blood is exchanged for O2. The oxygen-rich blood is carried back to the heart through the pulmonary veins.

2.
3.

4.

This oxygen-rich blood is then pumped from the heart to the many tissues and organs of the body, through the systemic arteries. In the tissues, the arteries narrow to tiny capillaries. Here, O2 in the blood is exchanged for CO2. The capillaries widen into the systemic veins, which carry the carbon-dioxide-rich blood back to the heart.

5.

6.

Figure 7 This is a schematic diagram of the flow of blood through the circulatory system, showing the sites of O2/CO2 exchange in the body. Note: The components of this diagram are not drawn to scale. How do CO2 and H+ promote the release of O2 from hemoglobin? These species help form interactions between amino-acid residues at the interfaces of the four subunits in hemoglobin. These interactions are called "salt bridges," because they are between positively-charged and negatively-charged amino-acid residues on different subunits of the same protein (Figure 8). When "salt bridges" form, the subunits are held in a position that "tugs on" the histidine that is attached to the heme iron. (See Figure 5.) This favors the domed configuration, which is the deoxygenated form of hemoglobin.

Figure 8 On the left is a schematic diagram of the interface of two subunits of the deoxygenated hemoglobin protein. In the presence of CO2 and H+ (e.g., in the muscles), charged groups are formed on the amino acid residues lining the subunit interface. These charged groups are held together by ionic interactions, forming "salt bridges" between the two subunits, and stabilizing the deoxygenated form of hemoglobin. When blood passes through the alveolar capillaries of the lungs, CO2 and H+ are removed from the hemoglobin, and the oxygenated configuration is favored (right). Note: The components of this diagram are not drawn to scale. When the concentration of protons (H+) is low (pH 9), positive charges do not form on the residues at the subunit interfaces, so the salt bridges cannot form (right image in Figure 8). However, at pH 7, histidine residues at the subunit interfaces (not the histidine residues that bind the heme groups) can accept an additional proton (H+), and hence become positively charged (Equation 1). When salt bridges form by the interaction of these interfacial histidine residues and nearby negatively-charged amino-acid residues, the deoxygenated hemoglobin structure is favored, and oxygen is released (left image in Figure 8).

(1

The number of negatively-charged residues in the salt bridges is increased in the presence of carbon dioxide. CO2 binds to the amino (-NH2) group of certain amino acid residues at the subunit interfaces to produce a negatively-charged group (-NHCOO-) on the residue (Equation 2). This negatively-charged group can form salt bridges with the positive charges on the protonated histidines described above. The H+ produced by Equation 2 can also be accepted by histidine residues at the subunit interfaces (via Equation 1).

Thus, hemoglobin's biological function is regulated by the changing of the overall protein structure. This structure is altered by the binding or releasing of CO2 and H+ to the interfaces of the subunits in hemoglobin (Figure 8).

Questions on the Bohr Effect:

Does CO2 bind at the same site on the hemoglobin molecule as O2? If not, where does CO2 bind? In muscles, the oxygen released by hemoglobin is taken up by myoglobin. Myoglobin is a muscular protein that stores oxygen and allows it to diffuse throughout the muscle fibers so that it can be used by the muscle. Myoglobin contains only one subunit (and thus only one heme group), which is very similar to one of the subunits of hemoglobin. Would you expect myoglobin to exhibit the Bohr effect (i.e., would myoglobin release O2 in the presence of CO2 and H+)? Briefly, explain your reasoning.

Summary Blood is an amazing and vitally important part of the body, because it contains many finely-tuned chemical systems that allow it to maintain the chemical environment needed for the body's metabolism. One of the most important functions of blood is delivering O2 to all parts of the body by the hemoglobin protein. O2 is carried in the hemoglobin protein by the heme group. The heme group (a component of the hemoglobin protein) is a metal complex, with iron as the central metal atom, that can bind or release molecular oxygen. Both the hemoglobin protein and the heme group undergo conformational changes upon oxygenation and deoxygenation. When one heme group becomes oxygenated, the shape of hemoglobin changes in such a way as to make it easier for the other three heme groups in the protein to become oxygenated, as well. This feature helps the protein to pick up oxygen more efficiently as the blood travels through the lungs. Hemoglobin also enables the body to eliminate CO2, which is generated as a waste product, via gas exchange in the blood (CO2 exchanged for O2 in the lungs, and O2 exchanged for CO2 in the muscles). The species generated as waste by the oxygen-consuming cells actually help to promote the release of O2 from hemoglobin when it is most needed by the cells. Hence, hemoglobin is a beautiful example of the finely tuned chemical systems that enable the blood to distribute necessary molecules to cells throughout the body, and remove waste products from those cells.

CHIME Files To view the molecules interactively, please use Chime. To download the pdb files for viewing and rotating the molecules shown above, please click on the appropriate name below or on the "interactive" button below each molecular-model figure in the text.

Alpha-helix structure (alphahelix.pdb) One subunit from Hemoglobin (subunit.pdb) Hemoglobin (hemoglobin.pdb) Heme group bound to histidine residue (heme.pdb)

Additional Links:

Naming Coordination Compounds summarizes the steps and provides examples of naming compounds containing coordination complexes. For additional information on hemoglobin, see the hemoglobin tutorial by Eric Martz of the University of Massachusetts.

Note: You need CHIME to view this tutorial. You can download Chemscape Chime here.

To learn about porphyrin and other chelating agents, see this "Chemical of the Week" site from the University of Wisconsin. Another porphyrin molecule, similar to heme but containing Mg rather than Fe as the central atom, is chlorophyll. You can learn about the structure , function, and spectroscopy of chlorophyll from this site. (Note: You will be studying chlorophyll in lab at the beginning of Chemistry 152.)

References: Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm. Ji, X. et al. "Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin," (1996) Biochemistry, 35, 3418. Hemoglobin PDB coordinates, Brookhaven Protein Data Bank.

Kavanaugh, J.S. et al. "High-resolution x-ray study of deoxyhemoglobin Rothschild 37beta trp->arg: a mutation that creates an intersubunit chloride-binding site," (1992) Biochemistry, 31, 4111. Deoxyhemoglobin PDB coordinates, Brookhaven Protein Data Bank. Kilmartin, J.V. "Interaction of haemoglobin with protons, CO2, and 2,3-diphosphoglycerate," (1976) Br. Med. Bull., 32, 209. Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org. Royer Jr., W.E. "High-resolution crystallographic analysis of co-operative dimeric hemoglobin," J. Mol. Biol., 235, 657. Oxyhemoglobin PDB coordinates, Brookhaven Protein Data Bank. Stryer, L. Biochemistry, 4th ed. W.H. Freeman and Co., New York, 1995, p. 154-168.

Blood Groups, Blood Typing and Blood Transfusions The discovery of blood groups Experiments with blood transfusions, the transfer of blood or blood components into a person's blood stream, have been carried out for hundreds of years. Many patients have died and it was not until 1901, when the Austrian Karl Landsteiner discovered human blood groups, that blood transfusions became safer. Mixing blood from two individuals can lead to blood clumping or agglutination. The clumped red cells can crack and cause toxic reactions. This can have fatal consequences. Karl Landsteiner discovered that blood clumping was an immunological reaction which occurs when the receiver of a blood transfusion has antibodies against the donor blood cells. Karl Landsteiner's work made it possible to determine blood groups and thus paved the way for blood transfusions to be carried out safely. For this discovery he was awarded the Nobel Prize in Physiology or Medicine in 1930.

What is blood made up of? An adult human has about 46 liters of blood circulating in the body. Among other things, blood transports oxygen to various parts of the body. Blood consists of several types of cells floating around in a fluid called plasma. The red blood cells contain hemoglobin, a protein that binds oxygen. Red blood cells transport oxygen to, and remove carbon dioxide from, the body tissues. The white blood cells fight infection. The platelets help the blood to clot, if you get a wound for example. The plasma contains salts and various kinds of proteins.

What are the different blood groups?

The differences in human blood are due to the presence or absence of certain protein molecules called antigens and antibodies. The antigens are located on the surface of the red blood cells and the antibodies are in the blood plasma. Individuals have different types and combinations of these molecules. The blood group you belong to depends on what you have inherited from your parents. There are more than 20 genetically determined blood group systems known today, but the AB0 and Rh systems are the most important ones used for blood transfusions. Not all blood groups are compatible with each other. Mixing incompatible blood groups leads to blood clumping or agglutination, which is dangerous for individuals. Nobel Laureate Karl Landsteiner was involved in the discovery of both the AB0 and Rh blood groups.

AB0 blood grouping system According to the AB0 blood group system there are four different kinds of blood groups: A, B, AB or 0 (null).

Blood group A If you belong to the blood group A, you have A antigens on the surface of your red blood cells and B antibodies in your blood plasma.

Blood group B If you belong to the blood group B, you have B antigens on the surface of your red blood cells and A antibodies in your blood plasma.

Blood group AB If you belong to the blood group AB, you have both A and B antigens on the surface of your red blood cells and no A or B antibodies at all in your blood plasma.

Blood group 0 If you belong to the blood group 0 (null), you have neither A or B antigens on the surface of your red blood cells but you have both A and B antibodies in your blood plasma.

Rh factor blood grouping system Many people also have a so called Rh factor on the red blood cell's surface. This is also an antigen and those who have it are called Rh+. Those who haven't are called Rh-. A person with Rh- blood does not have Rh antibodies naturally in the blood plasma (as one can have A or B antibodies, for instance). But a person with Rh- blood can develop Rh antibodies in the blood plasma if he or she receives blood from a person with Rh+ blood, whose Rh antigens can trigger the production of Rh antibodies. A person with Rh+ blood can receive blood from a person with Rh- blood without any problems.

Blood group notation According to above blood grouping systems, you can belong to either of following 8 blood groups: A Rh+ A RhB Rh+ B RhAB Rh+ AB Rh0 Rh+ 0 Rh-

Do you know which blood group you belong to?

Blood typing how do you find out to which blood group someone belongs? 1. You mix the blood with three different reagents including either of the three different antibodies, A, B or Rh antibodies. 2. Then you take a look at what has happened. In which mixtures has agglutination occurred? The agglutination indicates that the blood has reacted with a certain antibody and therefore is not compatible with blood containing that kind of antibody. If the blood does not agglutinate, it indicates that the blood does not have the antigens binding the special antibody in the reagent. 3. If you know which antigens are in the person's blood, it's easy to figure out which blood group he or she belongs to!

A person with A+ blood receives B+ blood. The B antibodies (yellow) in the A+ blood attack the foreign red blood cells by binding to them. The B antibodies in the A+ blood bind the antigens in the B+ blood and agglutination occurs. This is dangerous because the agglutinated red blood cells break after a while and their contents leak out and become toxic.

What happens when blood clumps or agglutinates? For a blood transfusion to be successful, AB0 and Rh blood groups must be compatible between the donor blood and the patient blood. If they are not, the red blood cells from the donated blood will clump or agglutinate. The agglutinated red cells can clog blood vessels and stop the circulation of the blood to various parts of the body. The agglutinated red blood cells also crack and its contents leak out in the body. The red blood cells contain hemoglobin which becomes toxic when outside the cell. This can have fatal consequences for the patient. The A antigen and the A antibodies can bind to each other in the same way that the B antigens can bind to the B antibodies. This is what would happen if, for instance, a B blood person receives blood from an A blood person. The red blood cells will be linked

together, like bunches of grapes, by the antibodies. As mentioned earlier, this clumping could lead to death.

Blood transfusions who can receive blood from whom? Of course you can always give A blood to persons with blood group A, B blood to a person with blood group B and so on. But in some cases you can receive blood with another type of blood group, or donate blood to a person with another kind of blood group. The transfusion will work if a person who is going to receive blood has a blood group that doesn't have any antibodies against the donor blood's antigens. But if a person who is going to receive blood has antibodies matching the donor blood's antigens, the red blood cells in the donated blood will clump. People with blood group 0 Rh are called "universal donors" and people with blood group AB Rh+ are called "universal receivers." Rh+ blood can never be given to someone with Rh - blood, but the other way around works. For example, 0 Rh+ blood can not be given to someone with the blood type AB Rh -. Blood Group AB Rh+ Antibodie s None Can give blood to AB Rh+ Can receive blood from AB Rh+ AB Rh A Rh+ A Rh B Rh+ B Rh 0 Rh+ 0 Rh AB Rh
-

Antigens

A, B and Rh

AB Rh

A and B

None AB Rh (Can AB Rh+ develop Rh antibodies) B A Rh+ AB Rh+

A Rh+

A and Rh

A Rh+ A Rh 0 Rh+ 0 Rh A Rh 0 Rh -

A Rh

B (Can develop Rh antibodies) A

A Rh A Rh+ AB Rh AB Rh+ B Rh+ AB Rh+

B Rh+

B and Rh

B Rh+ B Rh 0 Rh+ 0 RhB Rh 0 Rh -

B Rh

A (Can develop Rh antibodies) A and B

B RhB Rh+ AB RhAB Rh+ 0 Rh+ A Rh+ B Rh+ AB Rh+

0 Rh+

Rh

0 Rh+ 0 Rh -

0 Rh

None

A and B (Can develop Rh antibodies)

AB Rh+ AB Rh A Rh+ A Rh B Rh+ B Rh 0 Rh+ 0 Rh -

0 Rh