Clinical chemistry review sheet for mlt certification and ascp

A. CARBOHYDRATES

1. Describe and classify carbohydrates • Description

• Contain C, H and O molecules • Contain a C=O (ketone) and an –OH(aldehyde) functional group

• Classification

• Based on certain properties • The size of the base carbon chain • Location of the CO functional group • Number of sugar units • Stereochemistry of compound

• Chemical Properties

• Some ( not all ) carbs are reducing substances (donate electrons) • Chemical reduction of other substances • These sugars must contain an aldehyde or ketone group • Reducing sugars

o Glucose o Maltose o Lactose o Fructose o Galactose

• Sucrose is not a reducing substance

2. Describe carbohydrate metabolism • Glucose is primary energy source • Nervous tissue cannot concentrate or store carbohydrates, so a steady supply of

glucose is needed • Once the level of glucose falls below a certain range, normal function is impaired • Carbohydrate Breakdown Ultimate Goal

o Convert glucose to CO2 and water with ATP as a by-product

3. Discuss glycolysis as it pertains to carbohydrate metabolism and carbohydrate detection methods

• Hydrolysis of glucose by an enzyme into pyruvate or lactate; • This process is anaerobic

4. Fasting blood glucose levels • Hyperglycemic

o Fasting blood glucose > 100 mg/dL • Hypoglycemic

o Fasting blood glucose < 50 mg/dL

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5. Describe glycolysis • Glycolysis – the conversion of glucose and other hexoses into lactate or pyruvate • Breakdown of glucose for energy production

6. Describe carbohydrate breakdown

• Ultimate Goal o Convert glucose to CO2 and water with ATP as a by-product o Possible channels o Converted to liver glycogen and stored o Metabolized to CO2 and H2O o Converted to keto-acids, amino acids, and proteins o Converted to fats and stored in adipose tissue

• Biochemical pathways in carbohydrate breakdown o Embden-Meyerhoff pathway o Converts glucose to pyruvate/lactate o Primary energy source for humans o Hexose monophosphate shunt o Oxidizes glucose to ribose and CO2 o Produces NADPH as an energy source o Glycogenesis o Converts glucose to glycogen

7. Describe the role of the liver in maintenance of glucose levels.

• The liver maintains the glucose levels by: o Glycogenesis

Converts glucose to glycogen

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o Glycogenolysis – Breakdown of glycogen to form glucose Glycogenolysis occurs when plasma glucose is decreased Occurs quickly if additional glucose is needed Controlled by hormones & enzymes

o Gluconeogenesis Formation of glucose from non-carbohydrate sources, such as

amino acids, glycerol & fatty acids into glucose • Occurs mainly in the liver During long fasts, gluconeogenesis is required to maintain blood

glucose levels because glycogen stores are up in about 24 hours

• During a fast, the blood glucose level is kept constant by mobilizing the glycogen

stores in the liver

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8. What hormones does the liver use to maintain glucose levels?

• Insulin o Produced by the beta cells of the islets of Langerhans in the pancreas o Promotes the entry of glucose into liver, muscle, and adipose tissue to be

stored as glycogen and fat; o Inhibits the release of glucose from the liver o Insulin secretion controlled by:

Blood glucose level Certain Amino Acids ie. leucine, & arginine

• Glucagon o Secreted by the alpha cells of the pancreatic islets of Langerhans o Increases blood glucose by stimulating glycogenolysis and gluconeogenesis o 2nd most important glucose regulatory hormone o Referred to as a hyperglycemic agent o Synthesized in alpha cells of the islets of Langerhans o Action/Effect of

o Stimuli – decreased plasma glucose o Action Increases glycogenolysis & gluconeogenesis Promotes breakdown of fatty acids Promotes breakdown of proteins to form amino acids Increases plasma glucose concentration

• Somatostatin o Origin-Delta cells of the islets of Langerhans in the pancreas o Effect - increase plasma glucose o Actions

antagonistic to insulin,

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inhibits endocrine hormones including glucagon & growth hormone o Inhibits secretion of insulin, glucagon, and growth hormone resulting in an

increase in plasma glucose levels

• Other regulatory hormones o Epinephrine

One of two glucose regulating hormones from the adrenal gland Origin – adrenal medulla Action/effect

• Inhibits insulin secretion & release • Promotes lipolysis • Stimulates glycogenolysis • Immediate release of glucose

Stimuli • Neurogenic - based on physical / emotional stress. • Adrenal tumors

o Glucocorticoids - such as cortisol Origin – adrenal cortex Effect – antagonistic to insulin

• increases blood glucose • promotes gluconeogenesis from breakdown of proteins • inhibits the entry of glucose into muscle cells

Stimuli – anterior pituitary’s ACTH o Growth Hormone (GH) and Adrenocorticotropic Hormone (ACTH)

Origin – anterior pituitary gland Effect – antagonistic to insulin

• Increases plasma glucose levels • inhibits insulin secretion • inhibits entry of glucose into muscle cells • inhibits glycolysis • inhibits formation of triglycerides from glucose

Stimuli • decreased glucose stimulates its release • increased glucose inhibits its release

o Thyroid hormones (such as thyroxine) Origin – thyroid gland Effect

• Increases absorption of glucose from intestines • Promotes conversion of liver glycogen to glucose

o Stimuli – pituitary gland’s TSH

9. What hormones does the pancreas produce that regulate carbohydrate metabolism?

• Insulin

o Produced by the beta cells of the islets of Langerhans in the pancreas o Promotes the entry of glucose into liver, muscle, and adipose tissue to be

stored as glycogen and fat;

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o Inhibits the release of glucose from the liver • Glycagon

o Secreted by the alpha cells of the pancreatic islets of Langerhans o Increases blood glucose by stimulating glycogenolysis and gluconeogenesis

• Somatostatin o Synthesized by the delta cells of the pancreatic islets of Langerhans o Inhibits secretion of insulin, glucagon, and growth hormone resulting in an

increase in plasma glucose levels

10. What impact does cortisol, catecholamine hormones and thyroid hormones have on glucose levels.

• Cortisol

o Secreted by the adrenal glands; o Stimulates glycogenolysis, lipolysis, and gluconeogenesis o Increases plasma glucose by decreasing intestinal entry into cells and

increasing gluconeogenesis. • Epinephrine

o Increases plasma glucose by inhibiting insulin secretion. o Secreted by the medulla of the adrenal glands. o It stimulates glycogenolysis and lipolysis; o It inhibits secretion of insulin. o Physical or emotional stress causes increased secretion of

epinephrine and an immediate increase in blood glucose levels. • Thyroid hormone

o Secreted by the thyroid gland; o Stimulates glycogenolysis and gluconeogenesis; o Increases glucose absorption from the intestines

11. Describe the metabolic defect in Diabetes Mellitus?

• Glucose does not get into the cells

12. What are typical glucose levels, insulin levels, and ketone levels in this disease?

1) Type I Diabetes Mellitus- o Glucose levels are increased o Insulin levels are decreased o Ketones present

2) Type II Diabetes Mellitus

o Glucose levels are increased o Insulin levels are normal to decreased o Glucagon response is decreased o No ketones present.

13. What is glycosylated hemoglobin?

• Glycated hemoglobin is formed from the nonenzymatic, irreversible attachment of glucose to hemoglobin A1.

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• Measurement of glycated hemoglobin reflects blood glucose levels for the past 2– 3 months.

• It is useful in monitoring effectiveness of treatment and compliance of diabetic individual to treatment protocol.

• The primary determinant in the rate of hemoglobin A-1c synthesis is the life span of the Red Blood Cell and the level of average glucose concentration

14. What are the normal glucose levels in a fasting individual?

• Normal glucose level (fasting)- 70 to 110 mg/dl

15. What are panic or critical glucose values?

• Panic Values- >126 (fasting), > 200 (random or glucose tolerance test)

16. Describe the relationship between glucose levels in urine and serum.

• Glucose will never be found in urine unless the serum glucose is high enough to exceed the renal threshold and spill over into the urine

• Glucose is filtered by the glomeruli, reabsorbed by the tubules, and normally not present in urine.

• If the blood glucose level is elevated, glucose appears in the urine, a condition known as glucosuria.

• An individual’s renal threshold for glucose varies between 160 and 180 mg/ dL. • When blood glucose reaches this level or exceeds it, the renal tubular transport

mechanism becomes saturated, which causes glucose to be excreted into the urine.

17. Name inherited disorder of carbohydrate metabolism • Glucose 6 phosphatase deficiency AKA von Glerke • Galactosemia

o Characterized by a deficiency or absence of galactokinase, o Enzyme defect prevents metabolism of galactose. o Galactose is found in milk as a component of lactose, with galactosemia

generally identified in infants.

18. Why should serum for glucose be removed from the red cells as soon after collection as possible?

• Serum should be removed from RBC’s ASAP because the cells will use the

glucose and falsely decrease the glucose level.

19. What anticoagulant preservatives are used for glucose specimens? Why?

• Sodium fluoride is the anticoagulant of choice because it inhibits glycolytic enzymes.

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20. What is a two-hour post prandial glucose? Why is it performed?

• This is a sample taken two hours after eating and can determine how well the body is using the glucose.

• This sample will show insulin function which is the main reason it is performed.

21. Why are D-xylose tolerance tests performed?

• This test is done to differentiate malabsorption from pancreatic insufficiency.

22. What is the normal range of CSF glucose?

• Normal CSF glucose- 40-70 mg/dl • CSF glucose is 2/3 of the plasma glucose which is due to the glucose entering the

CSF by facilitative transport. • The carrier mechanism is responsible for transporting glucose across the downhill

gradient. • Meningitis can cause a change in the CSF glucose levels.

23. Describe states that result in alteration of serum glucose.

• Diabetes mellitus Increases glucose (insulin is not working) • Pancreatitis- Increases glucose (insulin not produced) • Recent meal intake Increases glucose • Fasting Decreases glucose

24. Lactate • The normal end product of glucose metabolism is pyruvate; • Lactate is produced under conditions of oxygen deficit (anaerobic metabolism). • The production and accumulation of lactate in the blood and its measurement aid in

assessing the degree of oxygen deprivation that is occurring. • Change in the blood lactate level precedes a change in blood pH. • Lactate is metabolized by the liver via gluconeogenesis.

25. What are normal glycosylated hemoglobin ranges?

• Normal glycosylated hemoglobin range o 4.5-8.0

• High values indicate that the patient has not been following the proper diet

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B. LIPIDS

1. Describe the cholesterol • Function

o Used to manufacture and repair cell membranes, o Used in synthesis of bile acids and vitamin D

• Synthesized in the liver and obtained from the diet. • Precursor for synthesis of bile acids, steroid hormones, and vitamin D • Transport mechanism

o Transported through the blood by LDL (low density lipoproteins) • Storage sites

o Stored in the skin, adipose tissue, and muscle cells. • Esterified cholesterol-2/3 of the total cholesterol is esterified

2. Describe triglycerides • Formed from one glycerol molecule with three fatty acid molecules attached via

ester bonds • Comprises 95% of all fats stored in adipose tissue • Transport mechanism

• Transported through the body by chylomicrons and VLDL

3. HDL-cholesterol • Synthesized by the liver and by the intestine • In normal lipid metabolism, HDL removes excess cholesterol from peripheral

tissues and transports it to other catabolic sites providing an antiatherogenic effect..

4. What is the role of lipase? • Lipase and bile acids are used to break down fats in lipid absorption. • Found in pancreas, with lesser amounts in gastric mucosa, intestinal mucosa,

adipose tissue • Clinical significance:

o Increased serum levels in acute pancreatitis occur in 4– 8 hours after the onset of pain, with peak values in 24 hours, and return to normal in 8– 14 days.

5. List the bile acids • Cholic acid, • Glycocholic acid • Taurocholic acid

6. Where are bile acids synthesized? • Liver

7. What does the presence of bile acids in serum indicate? • . The presence of bile acids in serum can indicate liver disease.

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8. What are chylomicrons? • Triglycerides are transported through the body by chylomicrons • Chylomicrons- the largest lipoprotein particles with diameters ranging from 80-1200

nm. They are 90-95% triglycerides, 2-6% phospholipids, 2-4% cholesteryl ester, 1% free cholesterol, and 1-2% apolipoprotein.

•

9. How does serum appear with increased chylomicrons? increased cholesterol? increased triglycerides? • Increased Chylomicrons- a creamy layer appears on top of the serum. • Increased Cholesterol- serum appears milky white • Increased Triglycerides- serum appears turbid.

10. Given a value of 38 mg/dl for the HDL, 140 for triglycerides and 210 for total cholesterol, calculate the LDL and VLDL.

• VLDL= triglycerides/5 • LDL= total cholesterol- VLDL- HDL • VLDL= 140/5 =28 • LDL= 210-28-38= 144

C. LIVER FUNCTION AND HEME-DERIVATIVES

1. Describe liver function in reference to each of the following:

3) Carbohydrate synthesis and metabolism • The liver uses glycogenesis to make glycogen from glucose, simple sugars

and amino acids. • The glycogen produced is stored and used as needed. • The liver also uses glucose for maintenance of mitochondrial NADH and to

generate ATP in the Embden Myerhoff pathway. 4) Protein synthesis and metabolism

• The liver makes various proteins (transferrin, prothrombin, and ceruloplasmin) which are used to transport materials like iron, and copper.

• The liver also uses the proteins for nutrition, to regulate oncotic blood pressure (albumin), and for coagulation.

o These proteins include albumin, HDL, LDL, VLDL, haptoglobin, angiotensin, erythropoiten, and many others.

o These proteins also are used for nitrogen excretion by processing ammonia, urea, creatinine and uric acid.

5) Lipid synthesis and metabolism- • This is where cholesterol, triglycerides and phospholipids are synthesized. • Free fatty acids are metabolized in the citric acid cycle into NADH, and bile

acids are produced form cholesterol. 6) Porphyrin synthesis

• The liver produces the enzyme aminolevulinic acid synthase which controls the synthesis of porphyrins which eventually form heme molecules.

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7) Bile acid synthesis • the bile acids are conjugated with amino acids to form bile salt. • It is synthesized by cholesterol in the bile ducts and ends up in the intestine

where the lipids are digested. 8) iron and vitamin storage

• Iron is stored in the liver and transported wherever needed by transferrin, and vitamins are stored in the liver and available to be used whenever needed.

9) Excretion of metabolic end product and detoxification • It converts ammonia to urea.

10) Bile pigment formation • Bilirubin is the principal pigment in bile and is derived from the breakdown of

hemoglobin when aged red blood cells are phagocytized by the reticuloendothelial system, primarily in the spleen, liver and bone marrow.

2. Where is bilirubin produced • Bilirubin is produced in the reticuloendothelial system from the breakdown of

hemoglobin from senescent red blood cells • Bilirubin forms a complex with albumin for transport to the liver.

• In this form, bilirubin is unconjugated and not water soluble.

3. Describe heme catabolism by the reticuloendothelial system (extravascular hemolysis). List states associated with increased extravascular hemolysis.

• Hemoglobin is broken down extravascularly into globin and heme (iron and protoporphrin IX)

• Protoporphrin breaks down further into unconjugated bilirubin which is carried to the liver by albumin and conjugated with glucuronyl transferase.

o This is associated with RBC membrane defects and defects in the heme structure.

4. Identify the function of haptoglobin, hemopexin albumin and methemalbumin in heme catabolism in intravascular hemolysis. • Haptoglobin binds free hemoglobin and takes it back to the liver so it is not lost in

the urine, • Hemopexin albumin removes circulating heme from the blood and delivers it to the

liver, • Methemalbumin is free heme oxidized and bound to albumin which is carried to the

liver and acts as storage that can be used until enough hemopexin is produced and made available to the liver.

5. List states associated with increased intravascular hemolysis. • Intravascular hemolysis is associated with increased immunologic processes,

mechanical injury, and toxins.

6. Differentiate between unconjugated and conjugated bilirubin • Conjugated bilirubin

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o Bilirubin is conjugated in the hepatocyte endoplasmic reticulum with glucuronic acid to form bilirubin diglucuronide ( conjugated bilirubin).

o The reaction is catalyzed by UDP o Conjugated bilirubin is water soluble. o Conjugated bilirubin is excreted into the bile for storage in the gallbladder,

secreted into the duodenum in response to gallbladder stimulation, and reduced by anaerobic bacteria in the intestine to urobilinogen.

o Some intestinal urobilinogen is reabsorbed; A portion returns to the liver and some enters the circulation for

excretion in the urine, whereas the remaining portion in the intestines is oxidized by anaerobic bacteria for excretion in the stool as urobilin.

o Urobilin is an orange- brown pigment that gives stool its characteristic color. • Unconjugated bilirubin

o Bilirubin is produced in the reticuloendothelial system from the breakdown of hemoglobin from senescent red blood cells

o Bilirubin forms a complex with albumin for transport to the liver. o In this form, bilirubin is unconjugated and not water soluble.

7. Describe the process of bilirubin conjugation. What is the role of glucuronyl transferase and where is this enzyme synthesized? What relationship exists between enzyme synthesis and neonatal physiologic jaundice? • Hemoglobin is broken down into portoporphrin which is converted into

unconjugated bilirubin which is bound to albumin and carried to the liver where it is converted to conjugated bilirubin by the enzyme glucuronyl transferase (synthesized in the liver).

8. What is Kernicterus? Why does it develop? • This is a serious newborn condition that occurs in the central nervous system

because of high bilirubin levels. • It is caused by an under developed blood brain barrier, and because newborns do

not produce enough glucuronyl transferase.

9. Describe excretion of bilirubin and resulting formation of urobilinogen? Why is urobilinogen normally present in urine and serum? • Conjugated bilirubin is taken to the intestines where bacteria convert it into

urobilinogen. • Urobilinogen is normally present in the urine because a small amount of it is filtered

back to the liver where it is recirculated and sent to the kidney where it is excreted in the urine.

10. Prehepatic jaundice • Prehepatic jaundice occurs when there is excessive erythrocyte destruction, as

seen in hemolytic anemias, spherocytosis, toxic conditions, hemolytic disease of the newborn caused by Rh or ABO incompatibility, etc

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• In these cases, the rate of hemolysis exceeds the liver’s ability to take up the bilirubin for conjugation.

• Prehepatic jaundice is characterized by an increased level of unconjugated bilirubin in the serum.

11. Hepatic jaundice • occurs when the liver cells malfunction and cannot take up, conjugate, or secrete

bilirubin. o Gilbert syndrome:

Defect in the ability of hepatocytes to take up bilirubin; due to transport problem of bilirubin from the sinusoidal membrane to the microsomal region; characterized by mild increase in serum level of unconjugated bilirubin

o Neonatal physiological jaundice: Level of UDP- glycuronyltransferase is low at birth; Takes several days for the liver to synthesize an adequate amount

of the enzyme to catalyze bilirubin conjugation; causes increased serum level of unconjugated bilirubin

12. Posthepatic jaundice • Occurs when an obstruction blocks the flow of bile into the intestines. • This is referred to as extrahepatic cholestasis and may be caused by gallstones

obstructing the common bile duct, neoplasms such as carcinoma of the ampulla of Vater or carcinoma of the pancreas, and inflammatory conditions such as acute cholangitis or acute pancreatitis.

• Posthepatic jaundice is characterized by: o Significantly increased level of conjugated bilirubin in serum, o Increased level of unconjugated bilirubin in serum, o Increased conjugated bilirubin in the urine, o Decreased urine and fecal urobilinogen o Stool that appears pale in color.

13. Why are bilirubin determinations performed on amniotic fluid?

D. B. Porphyrins and Heme Derivates

1. What are the porphyrias? • These are deficiencies in the enzyme production that can be acquired or inherited.

They result in increased production of one of the heme precursors to which they are intermediates.

2. What is the physiologic function of porphyrins. • The most important function of porphyrias are to chelate iron from heme.

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3. When are plasma hemoglobin levels increased and how are they measured? • The plasma hemoglobin levels are increased during thalessemias and

hemoglobinopathies. • They can be measured by cellulose acetate electrophoresis or citrate agar

electrophoresis.

4. What reagent is used for the detection of urobilinogen? When would urobilinogen levels be decreased? Increased? • Ehrlrich's reagent is most often used for urobilinogen detection • • Increased Urobilinogen levels

o Excess hemolysis, o Liver damage by hypoxia o Exposure to various toxic agents.

• Decreased Urobilinogen levels o Obstructive jaundice because there is a limited delivery of bilirubin to the

gut.

5. When would myoglobin be increased? How are serum myoglobin levels measured? • Myoglobin is increased when there is trauma to skeletal or cardiac muscle.

(myocardial infarction). • Serum myoglobin levels are measured by Electrophoresis.

E. PROTEINS AND NON-PROTEIN NITROGEN

1. What are the functions of proteins in normal physiology? • Proteins function as transport carriers for other substances. • They transport substances to the proper sites for absorption, modification, or other

utilization.

2. Salt fractionation- • The proteins are fractioned out by the use of salts. The salts decrease the water

available for hydration of the hydrophilic groups and cause precipitation of the globulins.

3. Zwitterion- • An ion that has both positive and negative regions of charge.

4. Zeta potential • This is the potential difference between the negative charges on the surface of the

red blood cell membrane and the cations in the aqueous medium. • Cations are divided into two groups, those that always move with the RBC and

those that can move freely in the medium. • The zeta potential is measured from the boundary of these two cations to the

negative charge on the membrane.

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5. Polypeptide • Amino acids that combine to form proteins which link together to form peptides. • Many peptides linked together form a polypeptide.

6. Oligoclonal banding • Electrophoretic pattern of CSF form patients with multiple sclerosis with distinct

bands in the globulin zone.

7. Briefly describe the Kjeldahl techniques for determination of protein and non-protein nitrogen. • In this method the serum proteins are precipitated with an organic acid. • The nonprotein nitrogen is removed with the supernatent. • The protein pellet is digested in H2SO4 with heat and a catalyst (cupric sulfate). • Potassium sulfate can also be used to improve the digestion. • The H2SO4 oxidizes the C, H, and S in protein into CO2, CO, H2O, and SO2. • The nitrogen in the protein is then converted to ammonium bisulfite which is

measured by adding alkali and distilling the ammonia into a standard boric acid solution.

• The ammonium borate formed is then titrated with a standard solution of HCL to determine the amount of nitrogen in the original protein solution.

8. What are the major causes of increased and decreased albumin? Increased and decreased globulins?

9. What is the theory of refractometry? • Refractometry- the velocity of light is changed as it passes the boundary between 2

transparent layers (air and water) causing light to be bent. • When solute is added to water the refractive index at 20*C of 1.33 is increased by an

amount proportional to the concentration of the solute in the solution.

10. What are major interfering substances in the determination of serum protein by refractometry?

• Interfering substances- Nonprotien solids (electrolytes, urea, and glucose)

11. Name three ways to separate albumins from globulins. • Electrophoresis, • Chromatography • Precipitation

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12. Discuss the reasons for determining spinal fluid protein and glucose.

13. What are normal values for spinal fluid protein and glucose?

14. What results are expected from spinal fluid in meningitis ? • In meningitis, encephalitis, and neurosyphilis there would be a decreased glucose

level with increased protein levels (IgG).

15. What results are expected from spinal fluid in encephalitis? • In meningitis, encephalitis, and neurosyphilis there would be a decreased glucose

level with increased protein levels (IgG).

16. What results are expected from spinal fluid in neurosyphilis? • In meningitis, encephalitis, and neurosyphilis there would be a decreased glucose

level with increased protein levels (IgG).

17. Discuss the BCG method for determining albumin? Why is the pH important? • BCG (Bromocresol Green) method for determining albumin is a dye binding

procedure where positively charged albumin is attracted to and binds to the anionic dye.

• Once bound to the albumin, the dye has a different absorption maximum than free dye.

• The amount of albumin can be quantitated by measuring the absorbance of the albumin-dye complex to which it is directly proportional.

• The pH must be adjusted on the solution to make the albumin positively charged so it will bind to the dye.

18. What is Biuret reagent? Explain its function in determination of total protein. What are the major interfering substances?

• The biuret reagent contains sodium potassium tartrate to complex cupric ions to prevent their precipitation in the alkaline solution, and potassium iodide which acts as an antioxidant.

• In this procedure small peptides react and the color of the chelate produced has a different shade that seen with larger peptides (color varies from pink to reddish violet and is measured at 510nm).

• Major interfering substances are any compound with 2 or more of the following groups NHCH2 , and NHCS.

19. What is a protein-free filtrate? List 3 precipitating reagents used. • A protein free filtrate removes proteins from whole blood, serum, urine, or other body

fluids by precipitation with a precipitant and then filtration or centrifugation. • Some precipitating reagents are:

o Tungstic acid o O-toluidine, o Horseradish peroxidase, o Molybdate

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o Trichloroacetic acid

20. What is Nessler's reagent? In what other reactions may it be used? • Nessler's reagent:

o Double iodide of mercury and potassium. o This reagent is used in the determination of non-protein nitrogens (NPN). o It is also used in Nessleration reactions of urea nitrogen.

21. What is an A/G ratio? How is it used diagnostically? • A/G ratio determines how much albumin is in the body compared to globulins. • You use it in correlation with Total protein to determine if albumin or globulins are

low.

F. Specific serum proteins

1. What is alpha-1 antitrypsin? Ceruloplasmin? Name and describe disease processes involved with each protein.

• Alpha-1 antitrypsin o Acute phase reactant o Main function

Neutralizes trypsin-like enzymes that can cause hydrolytic damage to structural protein.

o Disease process: Severe deficiency of this is associated with severe degenerative

emphysematous pulmonary disease. • Ceruloplasmin

o Copper containing glycoprotein that stores 90% of the total serum copper. o Disease process:

Low serum concentrations of copper are associated with Wilson’s disease.

2. What is the function of haptoglobin? • Haptoglobin functions to bind free Hemoglobin by the alpha chain.

3. Name three methods for measuring haptoglobin. • 3 methods to measure Haptoglobin:

o Starch gel electrophoresis, o Radial immunodiffusion, o Immunonephelometric methods.

4. When is haptoglobin decreased? Increased? • Increased Haptoglobin levels:

o Inflammatory conditions,

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o Burns, and o Nephrotic syndrome

• Decreased Haptoglobin levels: o Intravascular hemolysis, o Transfusion reactions, o HDN, o Mechanical breakdown of RBC's, o Athletic trauma.

5. Define Troponin • Troponin- is a complex of 3 proteins that bind to the thin filaments of striated muscle

(cardiac and skeletal) but are not present in smooth muscle.

6. List three isoforms that make up the troponin complex. o 3 isoforms:

TnT, TnI, TnC

7. Describe the advantages of troponin over CKMB and name the method currently available to measure cTnI. o Cardiac troponin I is highly specific for myocardial tissue, and because it does

not normally circulate in blood it is 13x more abundant in the myocardium than CKMB on a weight basis.

o cTnI is very sensitive and can indicate even a minor amount of cardiac necrosis. o The relative increase of cTnI is greater than that of CKMB. o cTnI can be measured by: o Immunoenzymetric assays using 2 monoclonal Ab's directed against different

epitopes on the protein.

G. Protein Electrophoresis

1. What is a monoclonal gammopathy? polyclonal gammopathy? • A monoclonal gammopathy is a sharp narrow band in the late beta or gamma region

that suggests a monoclonal M spike. • The M spike is a spike of one class of Ig’s that is possibly metastasizing or producing

clones of itself and suggests cancer, possibly multiple myeloma. • A polyclonal gammopathy is a broad gamma band that is increased. It looks

abnormal and is usually caused by an infection. It is made up of more than one serum protein being increased.

2. Where are the sites of synthesis for the following proteins: a. albumin- liver b. alpha-1 globulin- liver c. alpha-2 globulin- liver

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d. beta globulin- liver e. gamma globulins- (IgA, IgG, and IgM)- made by B cells that become plasma

cells.

3. Discuss the clinical picture of multiple myeloma. What are the expected results of electrophoretic patterns on serum and urine of a myeloma patient? • In multiple myeloma you would see:

o Bence jones proteins found in the urine (free kappa and lambda light chains)

o "M" spike in electrophoretic pattern

4. Briefly describe serum protein electrophoresis. • Serum electrophoresis

o Serum samples are applied to the cathode end of a support medium strip that is saturated with an alkaline buffer (8.6). The strip is connected to 2 electrodes and a current is passed. All serum proteins are negatively charged at the 8.6 pH so they migrate toward the anode end.

5. Name the five bands that occur in serum and list the major proteins that migrate in the five bands.

6. Diagram a normal pattern as they migrate from anode to cathode in a barbital buffer and label each peak.

7. What type of electrophoretic patterns can be expected in the following disease states? Explain the patterns and sketch them.

a. Multiple myeloma

b. Nephrotic syndrome

c. Liver disease

d. Chronic infection

e. Acute phase reaction

f. Malnutrition

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8. Describe CSF protein electrophoresis. • CSF protein electrophoresis is done with the same technique as serum

electrophoresis, except agarose gel is used most often because it is a high resolution technique.

9. What bands are normally seen in CSF protein electrophoresis • Oligoclonal bands are: distinct bands seen in the globulin zone and they are

associated with multiple sclerosis (90%), and Inflammatory infectious neurological disease.

10. What are oligoclonal bands and what disease process are they associated with? • pre-albumin, • albumin, • alpha-1 globulin (antitrypsin), • insignificant alpha-2 globulin, • Beta-1 zone (transferrin) • Beta-2 zone.

11. Describe protein electrophoresis patterns would appear in the following situations and explain why the pattern appears as it does.

a. Electrophoresis of plasma instead of serum

b. Serum containing alpha-feto protein

c. Fresh serum containing complement

d. Bisalbuminemia

e. Serum containing C-reactive protein

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H. Creatinine, Bun, Uric Acid

1. What is NPN? Which compounds comprise 50% of the total NPN's? • Non-protein nitrogen (NPN)- nitrogen containing compounds that remain in the blood

sample after the removal of protein constituents. • Urea is the compound that comprises 50% of the total NPN’s.

2. What is BUN? What is Azotemia? How is urea nitrogen converted to urea mathematically?

• Blood urea nitrogen (BUN)- nitrogen in the blood in the form of urea. This is a measurement that is used to analyze the urea level.

• Azotemia- an elevated level of urea in the blood. • Bun x 2.14 = urea

3. What is creatine? How is it measured? • Creatine- a compound found in muscle synthesized from several amino acids. It

combines with high energy phosphate to form creatine phosphate which functions as an energy compound.

• We can measure creatine by the Jaffe method which measures creatine based on analyzing the sample for creatinine before and after heating in acid solution. The heating converts the creatine to creatinine and the difference between the two samples is the creatine concentration.

4. Relate elevations in uric acid to the following disease states:

• Primary gout- this is caused by increases of uric acid which cause sodium urates to precipitate in the joints. This can be caused by overproduction of uric acid, drugs, and alcoholism.

• Secondary gout- this gout is formed as a secondary infection caused by a larger problem like leukemia.

• Leukemia- this causes the increased breakdown of cell nuclei caused by chemotherapy which causes the uric acid levels to increase.

• Polycythemia- this causes the increased breakdown of cell nuclei caused by chemotherapy, much like leukemia does, which also increases the uric acid levels to increase.

• Glomerular nephritis- in this disease the nephrons of the glomerulus are damaged in the kidney which causes poor filtration and increased levels of the uric acid.

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• Multiple myeloma- this disease is also treated with chemotherapy which breaks down the nuceli and causes the uric acid to increase much like leukemia and polycythemia.

5. Relate uric acid production to purine catabolism. • Uric acid is formed from the catabolism of purines like adenosine and guanine in the

liver. This uric acid is transported by the plasma from the liver to the kidney where it is filtered by the glomerulus into the proximal tubules where most is reabsorbed and only small amounts are secreted into the urine.

6. What is the clinical significance of BUN? (renal, prerenal, postrenal). What creatinine values are expected in these conditions?

• BUN- (direct urea measurement from serum or plasma) • It is used extensively in the determination of renal function.

BUN CREATINE BUN/CR RATIO Pre-renal- Increased Normal Increased Renal- Increased Increased Normal Post renal- Normal Increased Decreased

7. What are normal values for BUN and creatinine? What is the normal ratio of BUN to creatinine? When is the ratio altered?

• Normal BUN- 7-18mg/dl • Normal Creatinine- 0.5-1.2mg/dl • Normal BUN/Creatinine ratio- 10:1-20:1 • The ratio is altered in:

o Low protein uptake o Acute tubular necrosis o Severe liver disease

• Diagram and describe the Berthelot reaction for BUN. Berthelot Reaction for BUN-

o Urea is hydrolyzed with urease, and the ammonia ion formed is reacted with phenol and hypochlorite in alkaline medium to form indophenol. Nitroprusside is used to catalyze the reaction.

o Absorbance of dissociated indophenol (blue chromogen) is measured at 560nm.

o REACTION: NH4 + 5NaOCC+ 2 phenol◊ indophenol + 5NaCl + 5H2O

8. What is the purpose of the following reagents? o urease- used ot prepare the stock suspension

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o sodium nitroprusside- catalyzes the reaction o phenol- converted to indophenol o alkaline hypochloride- aids in conversion of phenol to indophenol.

9. What are some advantages and disadvantages of this method? What anticoagulant must be avoided when using any urease method?

o Advantages- can use serum, plasma, or urine. o Disadvantages- contamination of urine with bacteria is common and can

cause decreased urea and formation of ammonia.

10. What kidney functions do the following clearance tests measure? • inulin- Reference substance for measuring GFR (glomerular filtration rate) • creatinine- universely used in assessment of GFR. • urea- not useful in monitoring GFR, but serum urea may provide useful clinical

metabolic • information. • p-amino-hippurate- reference substance for the measurement of renal plasma flow.

11. Diagram and describe the Jaffe reaction for creatinine. What substances give false positive reaction?

• Jaffe reaction- o The reaction occurs between creatinine and the picrate ion formed in the

alkaline medium and a red-orange adduct develops. Teh observed rate of the hydroxyl ion concentrations over a broad range of picric acid concentrations. This is measured spectrophotometricaly at wavelengths of 485-520nm.

• Substances that give false positives are: • Protein, • Glucose, • Vitamin C (ascorbic acid), • Acetone, • cephalosporin

12. Why is creatinine preferred to urea for clearance tests? What data are necessary to calculate creatinine clearance? Write the formula. What are the normal values for creatinine clearance?

• Creatinine is more specific for kidney function than urea is. • Data necessary for calculating creatinine clearance:

o Urine volume, o Creatinine concentration. in urine, o Creatinine concentration in plasma

• Creatine Clearance = (Urine Cr x Urine volume)/ (Plasma Cr) • Normals: Male= 97-137 ml/min Females = 88-128 ml/min •

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13. A creatinine clearance was performed on a male patient 1.5m tall and weighing 65kg. His blood contained 2.5 mg/dl creatinine. The urine creatinine was 50 mg/dl and the urine volume was 300 ml/4hrs. What was the creatinine clearance for this man?

• Creatine Clearance = (50 x 300)/ 2.5 x 1.76/ 1.60 (body surface area *see chart) •

14. What is creatinine? What is the normal range? What single disease state is associated with elevated creatinine. • Creatinine is a compound formed when creatine or creatine phosphate

spontaneously loses water or phosphoric acid. • It is excreted into the plasma at a relatively constant rate in a given individual and

excreted in the urine. Its decrease is associated with renal dysfunction as in glomerulonephritis.

15. Diagram the reaction, list reagents used and describe the principle of the oxidation reduction method for uric acid using phosphotungstic acid. • This is the most common method used. • It is based on the oxidation of uric acid in a protein-free filtrate with subsequent

reduction of phosphotungstic acid to tungsten blue. • It uses Na carbonate to provide the alkaline pH necessary for the color

development. • The blue color produced can be intensified by adding cyanide or by keeping the

proper pH.

I. Miscellaneous Proteins

1. Describe the method for detecting phenolketonuria? What enzyme deficiency results in phenolketonuria?

• Phenolketonuria results from a total absence of or absence of activity of the enzyme phenylalanine hydrolase (AKA phenylalanine-4-mono-oxygenase).

• You can use the Guthrie test to detect PKU.

2. What are cryoglobulins? How are they measured? • Cryoglobulins are serum protein that precipitates at temperatures lower than body

temperature.

3. What is alpha-fetoprotein? What does its presence signify? How is it detected? • Alpha-fetoprotein is a globulin protein synthesized in the fetal yolk sac and then by

the parenchymal cells of the liver.

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• It is measured to determine if there is increased passage of fetal proteins into the amniotic fluid.

• We also measure the alpha-fetoprotein levels in association with spinabifiida, renal tube defects, and general fetal distress.

4. What is carcinoembryonic antigen? What types of tumors is it most frequently associated with? What types of methods are used to measure it? • Carcinoembryonic anitgens (CEA) are glycoproteins which are associated with

numerous cancers (colon, lung, pancreas, stomach, or breast tissue tumors)

5. For each of the following tumor markers describe the types of tumors they are most often associated with and how they are measured. • CA125- ovarian cancer. • PSA- (prostate specific antigen)- Prostate cancer

J. ENZYMES

1. (L) Give the substrates for the following: LD, CK, AST, ALT, GGT, CK-MB • LD Lactate

o Catalyzes oxidation of Lactate to Pyruvate and the reverse reaction of Pyruvate to Lactate

o ischemia, o myocarditis, o cardiac congestion;

• CK Creatinine o Catalyzes the reversible phosphorylation of ATP o Muscular dystrophy, o muscle malignancies, o heart disease, o thyroid disease, o CNS disease.

• AST Aspartate o Transfers amino acids o This is higher in neonates due to their immature liver o Liver disease (20-100 times normal in hepatitis), o carcinoma, o cirrhosis, o liver disease o heart disease, o muscle disease, o gallbladder disease, o AMI, o pulmonary embolism

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• ALT Alanine o Catalyzes the transfer of an amino group of alanine to alpha-ketoglutarate o Enzymatic-UV Monitoring o Liver disease, o carcinoma o gallbladder disease, o cirrhosis, o hepatotoxicity

• GGT Glutathione o Transfers gamma-glutamyl residue from gamma-glutamyl peptides to amino

acids, water, and other small peptides o Liver disease, o obstructions of the internal liver or gallbladder, o alcoholism, o pancreatic problems

• CK-MB Creatinine o Catalyzes the reversible phosphorylation of ATP o Myocardial problems

2. (L) Define: a. Isoenzyme-

o one of several forms in which an enzyme can exist in various tissues. o Although they are similar they can be separated from each other by special

chemical tests (electrophoresis) to give more specific information. b. Coenzyme-

o These are enzyme activators that are usually heat stable and of low molecular weight.

o When these are combined with an inactive protein called an apoenzyme they form an active compound or a complete enzyme called holoenzyme.

c. Catalyst- o Substance that speeds up the rate of a chemical reaction without itself being

permanently altered of used up in the reaction. o They are effective in small quantities and are not used up in the reaction.

They can be recovered unchanged. d. Activator

o Substance in the body that converts an inactive substance into an active agent.

o Example: the hydrogen ions on pepsinogen converting it to pepsin. e. Inhibitor-

o Chemical substance that stops the enzyme activity. f. Hydrolase-

o Enzyme that causes hydrolysis. These catalyze bond cleavage by the addition of water.

g. Oxidoreductase-

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h. Enzyme that removes electorns and their corresponding electrons. i. Transferase-

o These enzymes move chemical grouping from one compound to another.

3. Relate amylase and lipase activity to the following disease states: a. Acute pancreatitis- both increase in this. b. Malabsorption- increased in both. c. Chronic pancreatitis- increased in both d. Pancreatic carcinoma- increased in both e. Cystic fibrosis- increased in both because it leads to malabsorption

4. What are the sources of acid phosphate in the body? What are normal ranges for acid phosphatase in males and females?

o Acid phosphate is found in most tissues in the body like bone, bone marrow, liver, spleen, RBC’s, platelets, and in the highest concentration in the prostate gland of the male.

5. 8. (L) Explain the clinical significance of alkaline phosphatase in the following disease states:

a. Obstructive jaundice- increased ALP levels b. Parenchymal jaundice- increased ALP levels c. Paget's disease- increased ALP levels d. Hyperparathyroidism- increased ALP levels e. Pregnancy- increased ALP levels

6. List enzymes elevated in hemolysis? What enzyme might be depressed with refrigeration and freezing? • Enzymes elevated in hemolysis:

o CK, LD, AST (aspartate transferase), ACP (acid phosphatase), ALP (alkaline phosphatase), and LIPASE

• Enzymes depressed by refrigeration: o LD (occurs at 4*C within 24 hours), and LIPASE (occurs if stored at 4*C for

3 weeks)

7. What is cholinesterase? Why is it important in presurgery cases? • Cholinesterase: an enzyme found in RBC’s, lungs, spleen, nerve endings, and brain. It

is responsible for the prompt hydrolysis of acetylcholine released at the nerve endings to mediate transmission of the neural impulse across the synapse. The degradation of acetylcholine is necessary to the depolarization of the nerve so that it can be repolarized in the next conduction.

o This is important to measure cholinesterase in presurgery to determine the amount of succinyl dicholine (muscle relaxer) that can be given in surgery without complications. (You can only give the amount of succinyl dicholine to the patient that the patients’ cholinesterase can rid their body of.)

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8. What is the relationship of amylase and lipase in pancreatic disease? Why are both tests necessary in the monitoring of the disease? o Amylase and Lipase are both elevated in pancreatic disease. o You must monitor both amylase and lipase in pancreatic disease because:

Amylase is more sensitive, but less specific (b/c also found in other parts of the body)

Lipase is more specific, but found in small quantities (less sensitive b/c only found in the pancreas)

9. Discuss the following in relation to amylase activity: activators, pH, temperature. o Activatiors: Calcium and Chloride ions o pH- optimal is 6.9 - 7.0 o Temperature- optimal is 37-40*C

10. Discuss prostatic disease and acid phosphatase levels in serum. • Total activities of ACP may reach 40-50 times the normal in severe stages of

prostate cancer. If the carcinoma is highly localized to the prostate there may only be slight increases in ACP activity. In benign hypertrophy of prostate, enzyme levels are normal.

11. How is the L(+) tartrate utilized in the determination of acid phosphatase? • Tartrate inhibits the activity of non-prostatic ACP so that specificity is enhanced

when it is used.

12. Explain heat separation of alkaline phosphate isoenzymes. How does heat effect the liver fraction? bone? placenta? • Heat separation of alkaline phosphatase Isoenzymes ALP activity is determined by measuring ALP before and after heating serum at

56*C for 10 minutes. Placental ALP is the most heat stable followed by intestinal, liver, then bone. Placental ALP will resist heat denaturation at 65*C for 30 min. If the residual activity after heating is <20% of the total prior to heating then it is

bone phosphatase. If the residual activity after heating is >20% of the total prior to heating then it is

Liver phosphatase.

K. Enzyme Electrophoresis

1. List the CK isoenzymes. Describe the makeup of each fraction and organs associated with each fraction. How are isoenzymes separated? • CK-1 ( brain, brain subunits)- brain, prostate, uterus, bladdar, placenta • CK-2 (muscle, brain subunits)- heart muscle and skeletal muscle • CK-3 (muscle, muscle subunits)- sketal muscle and heart muscle

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2. How can hemolysis affect the LDH electrophoretic pattern? What is the clinical significance of this? • Hemolysis can cause LDH electrophoresis to have an LD-1 to LD-2 flip. • Using a hemolyzed sample would cause the results to have a LD-1 to LD-2 flip as

seen in cases of Myocardial Infarctions and Hemolytic anemia.

L. ELECTROLYTES AND TRACE ELEMENTS

1. For each of the following give normal range, panic values, categorized as anion or cation if applicable and categorize as intracellular or extracellular if applicable. • Potassium – Cation • Sodium – Cation • Calcium – Cation • Magnesium – Cation • Bicarbonate (HCO-

3) – Anion

• Chloride (Cl) - Anion

2. Describe the relationship between electrolyte balance and water balance. Include the roles of the kidney, hypothalamus, ADH and the renin-aldosterone system. • The electrolyte balance and water balance are directly related. • The plasma sodium concentration depends greatly on the intake and excretion of

water. • If the sodium increases it stimulates thirst which will increase the intake of water,

and the kidneys have the ability to conserve or excrete large amounts of sodium depending on the blood volume which is directly related to the water volume.

• The excretion of water is largely affected by the ADH (which is secreted from the hypothalamus) release in response to the increase in blood volume.

• The renin-aldosterone system acts in the kidney to increase the retention of sodium and increase the excretion of potassium which will eventually increase the blood pressure by using the electrolyte to balance the water level.

3. List anticoagulants of choice and the effect of hemolysis if any on the following ions: K, Na, Ca, Mg, HCO3, Cl, P04, Fe. • All electrolytes should be determined using serum or heparinized plasma.

o K- hemolysis increases K levels o Mg- hemolysis increases Mg levels o HCO3- hemolysis increases HCO3 levels o P04- hemolysis increases PO4 levels o Fe- hemolysis increases Fe levels

• o Na-hemolysis does not effect this significantly

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o Ca- hemolysis does not effect this significantly o Cl- hemolysis does not effect this significantly

4. Identify the major functions of sodium, chloride, bicarbonate, and potassium. • Sodium- this electrolyte largely determines the osmolality of the plasma. • Chloride- this electrolyte maintains the electrical balance by balancing the

sodium charge, and using the chloride shift with bicarbonate. • Bicarbonate-this electrolyte is used to maintain the acid base balance and buffer

the blood. • Potassium- this electrolyte regulates the neuromuscular excitability, contraction

of the heart, ICF volume, and hydrogen ion concentration (pH).

5. Define and list conditions associated with each of the following:

• Hyponatremia- this is decreased levels of sodium in the blood and is associated with the blood volume status. It results from sodium loss in excess of water loss.

• • Hypernatremia- elevated levels of sodium in the blood and is associated with

increased sodium concentration because of excess water loss. It can be caused by increased sodium intake or decreased water intake.

• • Hypokalemia- decreased levels of potassium in the blood and is associated with

GI or urinary loss of potassium , or with increased cellular use of potassium. This can be caused by vomiting, diarrhea, etc.

• • Hyperkalemia- increased levels of potassium in the blood and is associated with

diabetes mellitus, or metabolic acidosis.

6. What disease process results with increased bicarbonate? decreased bicarbonate? • Metabolic acidosis is related to the decrease in bicarbonate. • Metabolic alkalosis is related to the increase in bicarbonate.

7. Define titration. Explain its application to chloride methods. • Titration is the diluting out of a sample with a liquid reagent of a known strength

and measuring the volume necessary to convert the sample through a given reaction.

• In the chloride titration method the chloride ions combine with the mercuric ions to form soluble and undissociated mercuric chloride. The proteins in the serum are precipitated with the tungstic acid and an aliquot of the filtrate is titrated with an acidic solution of mercuric nitrate using a color indicator. This color indicator turns violet-blue at the first excess of mercuric ion.

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8. List disease processes in which hyperchloremia and hypochloremia occur but sodium is normal. • Hyperchloremia- this is an increase of serum chloride and occurs in situations

where there is an excess loss of bicarbonate ion due to GI losses, RTA, or metabolic acidosis.

• Hypochloremia-this is an decrease of serum chloride and occurs with the excess loss of chloride from prolonged vomiting, diabetic ketoacidosis, or aldosterone deficiency.

9. List causes of an increased anion gap? decreased anion gap? • Decreased anion gap is rare, but may be seen in multiple meyloma because of

abnormal proteins. It can also be caused by instrument error. • Increased anion gap- this may be caused by uremia, ketoacidosis (seen in

starvation or diabetes), posioning due to ingestion of substances like methanol or ethylene glycol, lactic acidosis, or severe dehydration which causes increased plasma proteins or instrument error.

10. Explain why stock standards of sodium and potassium are kept in plastic containers. • Stock standards of Na and K are kept in plastic containers because glass

containers leach out the Na and K from the sample.

11. What is an "anion gap". List formulas for its determination. • Anion gap- the difference between unmeasured anions and unmeasured cations.

It is useful for indicating an increase in one or more of the unmeasured anions in serum.

• AG = Na - (Cl + HCO3) • AG = (Na + K) - (Cl + HCO3)

12. Why is an anion gap routinely performed on all sets of electrolytes? What is an unacceptable gap? What is standard operating procedure when an anion gap is unacceptable? o An anion gap is routinely performed on all sets of electrolytes because it is useful

in indicating an increase in one or more of the unmeasured anions in serum, and for QC on th analyzer (an abnormal gap can indicate an analyzer problem if performed on a person in good health).

o An acceptable gap is 10-20:1, so greater than or less than that would be considered unacceptable and would need to be rerun.

13. Describe the impact of each of the following on serum potassium levels. • administration of insulin- decrease serum K levels (increases the cellular uptake

of K)

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• acidosis- increase serum K levels (excess H enters cell to be buffered and causes K to leave the cell to maintain electro neutrality)

• alkalosis- decreases the serum K (increases the cellular uptake of K)

14. Discuss the following factors influencing serum calcium and phosphorus levels: • parathyroid hormone- this hormone is used to increase the absorption of calcium

and increase the excretion of phosphorus. To increase calcium it breaks down the bone to release Ca (bone resprption), it conserves Ca by increasing the tubular reabsorption in the kidney and it stimulates the renal production of vitamin D which also increases the Ca absorption. To decrease phosphorus the blood concentration the PTH increases the renal excretion.

• calcitonin- this hormone is used to decrease calcium levels and increase the phosphorus levels which inhibits the actions of PTH and vitamin D.

• Vitamin D (calcitriol)-when the calcium is decreased or the phosphorus is increased this hormone is used to increase calcium by aiding the effects of PTH by causing more calcium to be stored or released. It decreases phosphorus by increasing the absorption of it in the intestines and increasing the reabsroption in the kidneys.

• plasma proteins-Albumin is the plasma protein that maintains the appropriate fluid in the tissues, and it binds various substances in the blood like calcium.

• serum pH- a decrease in pH will increase the phosphate levels in the serum like seen with antiacids.

15. Discuss calcium, phosphorus and PTH levels related to the following disease states: • bone disease-calcium will be normal to low, phosphorus will be normal to low,

and PTH will be normal to high. • malabsorption- calcium will be decreased, Phosphorus will be decreased, and

PTH will be increased. • renal failure- calcium will be low to normal, phosphorus will by high, and PTH will

also be high. • liver disease- calcium will be decreased, Phosphorus will be decreased, and PTH

will be increased. • primary hyperparathyroidism-Calcium will be Increased, Phosphorus will be

decreased, and PTH will be high. • secondary hyperparathyroidism-Calcium will be decreased, Phosphorus will be

low to normal to high, and PTH will be increased. • primary hypoparathyroidism-Calcium will be decreased, Phosphorus will be

increased, and PTH will be decreased. • secondary hypoparathyroidism-Calcium will be decreased, Phosphorus will be

increased or decreased, and PTH will be decreased.

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16. What is the physiologic relationship between calcium and phosphorus? Why? What are the physiologic functions of calcium and phosphorus? • Calcium and phosphorus are inversely related in the serum because phosphate

is an intracellular anion, and calcium is an extracellular cation.. • Calcium functions in bone matrix, as an enzyme activator, in coagulation and

complement, and in muscle contraction. • Phosphorus functions in production of ATP, GTP, CTP, UTP, and DNA

structures, as a major body buffer, and in the bone matrix.

17. What is the relationship of calcium levels to alkaline phosphatase activity? • Alkaline phosphatase levels are increased in periods of bone growth or

reconstruction which uses up the calcium and causes the serum levels of calcium to decrease. They both also function in bone matrix.

18. Describe three forms of body calcium. To which form is PTH most sensitive? How can only "active" calcium be measured? • Calcium forms- free-ionized Ca, protein bound calcium, and as complexed salts.

PTH is most sensitive to ionized calcium. To measure only the active calcium you must measure it under anaerobic conditions because an increase in pH can cause the protein bound Ca to increase which decreases the ionized Ca, and decreasing the pH can cause the protein bound Ca to decrease and the ionized Ca to increase.

19. Define and describe tetany? What are the relationship of magnesium and calcium to tetany. • Tetany- irregular muscle spasms. • Calcium- a rapid decrease in ionized calcium concentration will cause tetany. • Magnesium- this is required along with ATPase for normal Ca uptake following a

contration. It is also required for muscle cell stimulation by regulating the acetylcholine which is a potent neurotransmitter.

20. Describe the relationship between parathyroid hormone and magnesium levels. • The parathyroid hormone increases the renal absorption of magnesium and

enhanses the absorption of magnesium in the intestine. PTH regulates Ca, Na, and Mg.

21. What is the physiologic role of magnesium? Where is magnesium stored in the body?

o Magnesium functions as a cofactor for more than 300 enzymes including those important in glycolosis, transcellular ion transport, neuromuscular transmission, synthesis of carbohydrates and many others.

o Magnesium is stored in the bone (53%) and the rest (46%) is in the muscle, soft tissue and other organs.

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22. Discuss the following as they pertain to magnesium: o alcoholism- people who are alcoholics tend to have diets deficient in

magnesium or have problems with malabsorption (hypomagnesemia). o malabsorption- this causes a decrease in magnesium because it is not

absorbed (hypomagnesemia). o magnesium sulfate therapy- this is given parenterally to severely ill patients. o secondary hypoparathyroidism- this may cause an increased renal excretion

of magnesium due to an excess of calcium ions.

23. What is the physiologic function of iron? How is iron transported in the body? How is iron stored in the body?

• Iron functions as part of heme in hemoglobin. It is transported by transferrin and stored in the body as ferratin and hemosidrin.

24. Describe iron levels, %Saturation, TIBC, and ferratin levels in the following disease states:

• iron deficiency anemia-the % saturation is decreased, TIBC is increased, and ferratin is decreased.

• anemia of chronic infection- the % saturation is normal, TIBC is decreased, and ferratin is normal to increased.

• hemochromatosis- the % saturation is increased, TIBC is increased, and ferratin is increased.

25. What is the physiologic function of copper? What is Wilson's disease? ceruloplasmin? • Copper functions as an enzyme indicator, it acts on ferroxidase, and it acts it is a

component of enzymes or proteins involved in redox reactions. • Wilson’s disease- also known as hepatolenticular degeneration is a genetically

determined defect in ATPase where the copper is transported normally from the intestine into the liver, but cannot be transported from the liver into the bile.

26. What is the physiologic function of zinc? How is it measured? • Zinc functions as a cofactor for more than 300 enzymes. It can be measured by

atomic absorption.

27. Define: • total iron binding capacity- (amount of transferrin bound already) • An estimate of serum transferrin levels obtained by measuring the total iron binding

capability of a patients serum. Since transferrin represents most of the iron binding capacity of serum TIBC it is generally a good estimate of serum transferrin levels.

• % saturation- (transferrin saturation in the patients sample) • % saturation = total iron/TIBC x 100

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• unsaturated iron binding capacity- (the amount of sites available to bind iron in patients sample)

• UIBC = iron added – excess iron • Serum iron = TIBC – UIBC • latent iron binding capacity- (estimate of non-reacting iron bound to transferrin)

28. What is the source of blood ammonia? How is it detoxified? What disease processes are associated with increased ammonia? How is ammonia related to Reye's Syndrome?

• Blood ammonia arises or comes from the deamination of amina acids through the action of degestive and bacterial enzymes on proteins in the intestinal tract. Ammonia is released from metabolic reactions that occur in skeletal muscles during excercize.

• Ammonia is detoxified by the liver in the urea cycle where ammonia is converted to urea. • Hepatic failure, Reye’s syndrome, and urea cycle enzyme deficiencies are associated

with increased ammonia. • Reye’s syndrome is usually following a viral infection and it uses ammonia levels to

correlate with the severity of the disease and prognosis.

29. What is the purpose of renal synthesis of ammonia? • The kidney synthesizes ammonia to compensate for metabolic acidosis.

30. What are the three ketone bodies. When are they formed? List disease processes associated with increased ketones. o acetone, acetoacetic acid, and beta-hydroxybutyric acid. o These are formed as a product of incomplete fat metabolism, and are associated

with diabetes mellitus, starvation, and prolonged vomiting.

31. What is lactic acidosis? When does it occur? o Lactic acidosis- an increase or accumulation of lactic acid in the blood. o This occurs if there is improper oxidation of skeletal muscle and other tissues.

32. What role does hemoglobin play as an important buffer system in the body? What is carbonic anhydrase? What is chloride shift? • Hemoglobin buffers the blood by delivering oxygen to the tissues and then taking

the carbon dioxide to the lungs to be exhaled. • Carbonic anhydrase- this enzyme catalyzes the reaction of carbon dioxide to

bicarbonate and Hydrogen ion. • The chloride shift- the carbon dioxide goes into the RBC and forms carbonic acid,

this acid splits into hydrogen ion and bicarbonate. The bicarbonate leaves the cell and makes it more negative outside the cell, and more positive on the inside because of the hydrogen ion. At this point the chloride shifts into the cell to balance the electorneutrality of the cell.

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33. What are the main factors which influence the oxygen binding ability of hemoglobin? Specifically how do acidosis and alkalosis affect O2 saturation? • If there is increased oxygen then the hemoglobin binds more oxygen and if there is

less oxygen the hemoglobin picks up less oxygen and wants to hold on to it. The ability for hemoglobin to bind oxygen depends mostly on the availability of oxygen.

• Acidosis- the pH drops drastically and increases the hemoglobin affinity for O2. • Alkalosis- the pH is increased and it decreases the hemoglobin affinity for O2.

34. Why is heparin the anti-coagulant of choice for pH and blood gas work? How does it work?

• Heparin is used because it holds the ph constant in blood and prevents the change in gals levels in the sample.

35. Why are blood gas specimens placed in ice immediately after collection? • Blood gas specimens are placed on ice immediately after collection because: • The pH decreases with time if it is not placed on ice immediately after drawn. The lower

temperatures prevent the cells from undergoing glycolysis.

IX. ENDOCRINOLOGY

36. Give expected T4, T3 uptake, FTI and TSH levels in the following:

37. What is the function of the thyroid hormones in body metabolism? Describe the following disease processes:

• The thyroid hormone function is to stimulate the metabolism. Without the thyroid hormone the metabolism will decrease and the patient will experience obesity, mental retardation, edema (water in the tissues), decreased body temperature, and anemia.

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• cretinism- condition where the patient has a dysfunctional or no thyroid causing mental retardation and death. Autosomal recessive.

• juvenile myxedema-A dysfunction of the thyroid after birth that has very severe consequences. All 50 states require a T4 screening test for neonates.

• adult myxedema- A dysfunction of the thyroid later in life (>30 years) causing hair loss, dry skin, yellow pallor, thick tongue, and arterosclerosos.

• endemic goiter- an enlarged thyroid due to lack of iodine. • hashimoto's disease- the most common form of thyroid disease occurring mostly in

women 40to 60 years old. Treat with thyroxine. Autoimmune disease. • grave's disease- the patient has and AB to TSH receptor on the thyroid gland. This AB

causes the thyroid gland to think it is TSH and start to release T3 and T4 even though there is a decrease in actual TSH.

• thyroid tumors (goiters)- Tumors that cause the dysfunction of the thyroid gland allowing abnormal amounts of hormone to be released.

38. What are the roles of TSH? What amino acids are necessary for synthesis? • Body temperature stimulates the thyroid hormone which stimulates the

hypothalamus and causes it to release TRH to the pituitary gland. The TRH causes the pituitary gland to release TSH to stimulate the thyroid gland which then releases T3 and T4. TheT3 and T4 cause the body temperature to rise and act as the shunt to tell the hypothalamus to stop making the TRH and the pituitary to stop making TSH.

39. What is thyroglobulin? thyroid binding globulin? • Thyroglobulin- an iodine contaning protein secreted by the thyroid gland. • Thyroid binding globulin- A protein that binds thyroid hormones. The TBG assay is

used to confirm results of T3 and T4, or abnormalities it the relationship of the TT4 and T3U test. They can also be used as tumor markers for thyroid cancer.

40. Briefly describe each of the following groups synthesized by the adrenal cortex. What molecule is necessary for their synthesis? • glucocorticoids- (cortisol) these stimulate gluconeogenesis to increase glucose,

encourage the glycogen production and release from the liver, block epinephrine, act as and anti-inflammatory, inhibit WBC migration, phagocytosis, increase hematopoiesis and stabilize lysozome.

• mineralocorticoids- (aldosterone) these regulate potassium metabolism and regulate extracellular fluid volume. They also control water secretion by regulating Na absorption and K secretion.

• androgens- (testosterone, dehydroepiandrosterone, dehydroepiandosterone sulfate) These function in spermatogenesis and in the formation of the secondary male sex hormones.

• estrogens- (estrogen, estradiol, estriol) these function in ovulation, embryo preparation for implantation, and menstruation regulation

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41. Describe the feedback mechanism for cortisol. What is the function of cortisol? What is the role of ACTH? • The hypothalamus is stimulated by low glucose to secrete CRH which causes the

anterior pituitary to secrete ACTH to help make 11-deoxycortisol. Cortisol is formed and is found bound to transcortin or as free cortisol. The free cortisol stimulates he glucose production and when the need is met it acts as a shunt to tell the hypothalamus to stop making CRH.

42. Diagram the renin-angiotensin-aldosterone system. What is the purpose of this system? • The afferent arterioles secrete rennin when there is a decrease in blood pressure.

The rennin converts angiotensinogen to Angiotensin I which is converted to Angiotensin II. Angiotensin II stimulates aldosterone to be produced and secreted to increase the blood pressure by retaining sodium to passively hold water and increase the blood volume which increases the blood pressure.

43. What hormones make up the 17-hydroxysteriods? the 17-ketosteroids? Why are these tests performed? • Cortisone, cortisol, and 11-dehydroxycorticoids make up 17-hydroxysteroids. • Androgens make up the 17-keytosetriods. • These tests are performed to observe the levels for glucosteriods, testosterone,

and other androgens.

44. What are the catecholamines? Where are they synthesized? What is the precursor amino acid? What is their basic function?

• Catecholamines are epinephrine, norepinephrine, and dopamine. They are synthesized in the adrenal medulla of the adrenal glands. The precursor amino acid is tyrosine. Their basic function is to increase the blood pressure and heart rate when the body is excited in the fight or flight mechanism. They have the ability to break down adipose tissue and glycogen to be used for energy in the fight or flight mechanism.

45. Describe catecholamine levels in the following diseases: • Pheochromocytomas-Tumors of the adrenal medulla or the sympathetic ganglia of the

adrenal glands which cause overproduction of the catecholamines. • Infantile Neuroblastoma- Tumors of the adrenal medulla that cause an increase in

catecholamines.

46. What is the function of prolactin? When is it measured? • Prolactin acts to initiate lactation and maintain it in a pregnant female. It is

measured to evaluate pituitary tumors, galactorrhea, and fertility.

47. Describe levels of FSH and LH during follicular development, ovulation and the luteal phase? Why and how are these hormones measured? • FSH increases during follicular phase, goes down and rises again just after

ovulation, and drops slowly toward luteal phase.

Page 39

• LH is baseline in follicular phase, increases in ovulation, and decreases back to baseline during the luteal phase.

• These hormones are measured by RIA to assess pituitary and gonadal axis, fertility problems, to investigate puberty problems (late puberty), and to investigate pituitary tumors.

48. What is the function of gastrin? How is it related to Zollinger Ellison Syndrome? • Gastrin functions to secrete HCl to digest food, pepsin, intrinsic factor, pancreatic

hormones, and bile from the liver. • Zollinger Ellison (ulcers in the stomach or duodenum) syndrome has increased

Gastrin levels, so it can be separated from other peptic ulcers because they don’t increase in production of gastrin.

49. What is the function of the androgens? How are they measured? How is testosterone measured? • Androgens function in spermatogenesis and formation of secondary sex

characteristics of males. Androgens like testosterone are measured by RIA.

50. Briefly describe the function of the estrogens and progesterone. What test is used for total urinary estrogens? What is its principle? • Estrogen and progesterone both function in getting the uterus ready for embryo

implantation. • The Brown method (a colorimetric method using Kober reagent to look for a pink

color indicating that estriol is present) is used to measure total estrogen levels.

51. What is the primary site of synthesis of estradiol, and esterone? Explain why these two estrogens are measured? • Estradiol is produced in the ovary of a pre-menopausal woman. • Esterone is produced in the adrenal cortex of the adrenal glands of a post-

menopausal woman. • Both are measured to determine menstrual cycle disorders and ovulation in

women.

52. What does the T3 uptake measure? Why is this important? • T3 uptake measures- the # of free binding sites in the Thyroxine Binding Globulin. • This is important because it indirectly gives the amount of thyroxine in the serum

sample.

53. What is the free thyroxine index? T7? T12? How are they calculated? What is the importance of this calculation. • Free thyroxine index- estimates how much free T4 is in the blood. • T7 and T12- are pseudonyms for FTI (T7 = T3+ T4, and T12 = T3 x T4) • FTI- an index of thyroid status, it provides T4 and T3U which are useful in

diagnosing thyroid problems. • Free thyroxine index = (T4) x (% T3 uptake as a decimal)

Page 40

54. Discuss the purpose of each of the following steps in hormone determinations: • hydrolysis- remove and solubilize attachments (chemically or enzymatically) • purification- organic solvents purify the hormones • extraction- centrifugation, washing, and ion exchange • estimation- react, detect, and quantitate by various methods

55. What is diurnal variation? How does this impact cortisol levels and the collection of cortisol specimens? • Diurnal variation- levels of analytes rise and fall, they peak early in the day when

most of us are asleep. • Cortisol levels and ACTH (anterior pitutitary hormone, Cortocotropin) • They rise between 0600 and 0800 hours, then decrease all day long. At 2000 hrs

(8 pm), the level is 2/3 of what it is at 0800 hrs, so the best analysis is made form early morning specimens.

56. Describe Cushing's Syndrome and Addison's disease. Include their impact on diurnal variation, cortisol levels, glucose levels, aldosterone levels, electrolyte and water balance. How may secondary disease states be diagnosed? • Cushinn’s syndrome- Increased- cortisol, diurnal variation, aldosterone,

hypertension, and hyperglycemia (glucose), and decreased potassium. • Addison’s disease- Decreased- cortisol, diurnal variation, aldoserone, hypotension,

and hypoglycemia (glucose). Increased- potassium. • Addisons disease- Cosyntropin is given to th epatient which causes the cortisol

release, which aids in determining if patient has Addisons disease, because you look to see if cotrisol is released by the cortisol stimulating drug. If still no release of cortisol, it is a primary disease like addisons.

• Cushings disease- use hiht dexamethasome suppression test where there is a suppression of urine and plasma cortisol which only occurs in cushings disease.

57. When are plasma epinephrine and norepinephrine levels useful? • Epinephrine and Norepinephrine levels are useful in diagnosing stress, increased

catacholamines, decreased blood pressure, decreased blood volume, thyroid hormone deficiency, and congestive heart failure.

• Decreased catacholimines are found in hypotension.

58. When is it valuable to quantify HCG? What trimester of pregnancy are HCG levels used to monitor fetal health? • It is valuable to quantitate HCG to indicate how far along in pregnancy a patient is,

or if pregnant at all. Fetal health can be determined in the 1st and 3rd trimesters by looking at BHCG levels.

59. What is the function of serotonin? When does it increase? What is 5-hydroxy indolacetic acid? How is 5-HIAA measured? • Serotonin is released during coagulation by platelets and is involved in smooth

muscle stimulation and vasoconstriction. It is increased with carcinoid tumors that

Page 41

occur in the Ileum and appendix, 5-HIAA is a metabolite of serotonin and is excreted in the urine. It is measured colorimetrically after reacting it with 1-nitroso-2-napthol and nitrois acid (purple color).

60. Why are estriol levels measured? What trimester of pregnancy are these levels useful in? • Estriol levels are measured to help determine pregnancies. Levels in non pregnant

females can be measured in the ug’s, but pregnant women have levels in the mg range. These levels are useful in the third trimester (last 4-6 weeks of pregnancy)

M. TOXICOLOGY

1. List methods in which urines are screened for drugs of abuse? Why is urine preferred? What is the purpose of extraction? • You can use thin layer chromatography, gas chromatography, and immunoassays

to screen for drugs of abuse. Urine is used most often because the drugs are filtered through the kidneys and show up in the urine.

2. Why are chloramphenicol levels monitored? What disease process may result from chloramphenicol. • Chloramphenicol levels are monitored to make sure that ALA synthase is being

produced in enough quantity that heme synthase can be produced because chloramphenicol causes a decrease in ALA synthase, heme synthatase, and DNA synthase. This can be associated with disease processes like sideroblastic anemia.

3. What is the principle of the renish heavy metal test? What disease process is associated with lead poisoning? • The principal of the renisch heavy metal is to place a clean coiled copper wire in a

solution of 5-10 ml of gastric acid or urine with an equal amount of 2M HCL, then place in a hot water bath for 10 minutes, let sit one hour, and examine the copper wire for color change. ( blue or purple black- antimony; dull black- arsenic; shiny black- bismuth; and silver gray- mercury) Lead poisioning is associated with encephalopathy characterized by cerebral edema and hypoxia.

4. How is carbon monoxide poisoning usually detected? • This is usually detected by spot tests looking for carboxyhemoglobin (giving the

patient a cherry red appearance due to its color), or gas chromatography also looking for carboxyhemoglobin.

5. When does bromide toxicity result? How is it measured? How does bromide affect chloride determinations? • This toxicity results from organic and inorganic medication. It is measured by

immunoassay or thin layer chromotography, and it gives a false high in chloride determinations.

Page 42

6. What is the therapeutic usage of cyclosporine? methotrexate? • Cyclosporin- this is an immunosuppressive drug that is used to suppress host vs.

graft rejection of transplant organs. • Methotrexate- this is an antineoplastic drug that is used in therapy and involves the

rate of mitosis in normal cells versus neoplastic cells.

7. What are digoxin? digitoxin? Why are their levels so critical? • Digoxin- one of a group of cardiac glycosides obtained from digitalis plants which

restores the force of cardiac contraction in congestive heart failure. (drug used to treat cardiovascular problems)

• Digitoxin- another cardiac glycoside (less common than digoxin) that is used to treat cardiovascular problems.

• Critical levels- o Low- digoxin caused the atrium to be less electrically excitable o Moderate- reduce the rate of depolarization in the spontaneously

depolarizing conductive fibers. o High- diminishes the depolarization of the ventricular myocardium.

8. Define: • therapeutic range- concentration range of a drug which is beneficial to the patient

without being toxic. • peak level- one hour after the dose is given when the drug reaches peak

concentration in the body. • trough level- the lowest concentration of drug obtained in the blood, drawn

immediately prior to the next dose. • toxic value- drug levels outside of the therapeutic range. • bioavailability- tge fraction of a drug that is absorbed into the systemic circulation.

9. What is theophyline? When is it used? • A bronchodialator used to treat asthma or other chronic obstructive pulmonary

diseases

N. VITAMINS

1. What disease process is associated with decreased B12 and folic acid? What is the relationship between B12 and folic acid? • Pernicious anemia is associated with decreases B12 and folic acid. • In relationship between B12 an folic acid, B12 is used in the metabolism and

needed for the synthesis of folate which is needed for the production of nucleic acids (DNA)

Page 43

2. What is the function of Vitamin A? How is it related to beta-carotene? How are both measured? Why are serum beta carotene levels measured? • Vitamin A functions in growth, dim light vision, reproduction, immunity and mucous

secretion. Beta-carotene(pro-vitamin A) is the precursor to vitamin A and is composed of two moles of vitamin A.

• They are both measured by immunoassay or HPLC. • Beta carotene is measured in serum to indirectly quantitate 4. (P) Describe

the methods for measuring Vitamin B12? What is the function of cyanocobalamin?

Page 44

BLOOD GASES

Purpose Represents the acid/base status of entire body Provides information of lung function

Sample type

Whole Blood Arterial Sample – ABG

Preferred sample Sites are radial, femoral or brachial artery

Venous & Capillary Blood Can be used, but not preferred

Assessment performed STAT

SPECIMEN COLLECTION & HANDLING

Collected in heparinized plastic syringe (no air bubbles & no clots!!!) Often Collected by Respiratory Therapy

Collected anaerobically and put on ice. Ice

serves to slow cell metabolism.

Testing performed at 37o C, to emulate body temperature

PREANALYTICAL CONSIDERATIONS

Air bubbles Causes increases in pO2, pH Causes decreased in pCO2

Clots Can not run clotted whole blood on instrumentation

Glycolysis Cell respiration causes a decrease in pH, pO2

pCO2 increases Temperature

pH is temperature dependent. For every 1 degree rise in temperature, the pH decreases about 0.015 units

REFERENCE VALUES (ABG)

Component Arterial Blood Mixed Venous Blood

pH 7.35-7.45 7.31-7.41

pO2 80-100 mmHg 35-40 mmHg

O2 Saturation > 95% 70-75%

pCO2 35-45 mmHg 41-51 mmHg

HCO3- 22-26 mEq/L 22-26 mEq/L

Total CO2 23-27 mmol/L 23-27 mmol/L

Base excess -2 to +2 -2 to +2

INSTRUMENTATION

Electrochemistry Ion Selective Electrodes

Hemoglobin Concentration Spectrophotometry

DETERMINATION

Three components are directly measured pH pO2

pCO2

Values that can be calculated and reported include: Total CO2 or bicarbonate ion Base excess Oxygen saturation

PH MEASUREMENT

Measure of the hydrogen ion activity based on bicarbonate-carbonic acid buffer system

pH electrode has a thin membrane of glass separating two differing H+ concentrations, a H+ exchange occurs in the outer layers of the glass, causing a potential to develop.

A calomel half-cell or reference electrode is also immersed in the solution.

Both the pH and reference electrode are connected through a pH meter. The meter can measure voltage difference between the two and convert to pH units.

PO2 MEASUREMENT

Partial pressure of oxygen in the blood Measured by the O2 electrode to determine oxygen

content pO2 electrode or Clark electrode measures the

current that flows when a constant voltage is applied to the system

As dissolved O2 diffuses from the blood a change in current occurs which offers a direct pO2 measurement

PCO2 MEASUREMENT

Partial pressure of carbon dioxide in the blood pCO2 measured in mmHg x 0.03 indicates carbonic

acid (H2CO3)

pCO2 > 50 mmHg = HYPO ventilation

pCO2< 30 mmHg= HYPER ventilation

PCO2 MEASUREMENT

The pCO2 electrode or Severinghaus electrode consists of a pH electrode with a CO2 permeable membrane covering the glass surface. Between the two is a thin layer of dilute bicarbonate buffer.

Once the blood contacts the membrane and the CO2 diffuses into the buffer, the pH of the buffer is lowered

Change in pH is proportional to the concentration of dissolved CO2 in the blood

Siggaard-Anderson nomogram

CALCULATED PARAMETERS

Siggaard-Anderson nomogram Base Excess Total CO2 and bicarbonate concentration

BASE EXCESS

Determination of amount of base in the blood Determines the source of acid-base disturbance Base deficit usually indicates metabolic acidosis

Causes of:

Excess bicarbonate Deficit of bicarbonate

O2 SATURATION

Calculation/Derived Requires measured pH and pO2 values

Measured Requires a hgb measurement usually obtained by co-

oximetry Co-oximetry: measuring at multiple wavelengths to get light

absorption spectra

REFERENCES Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry:

Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

Carreiro-Lewandowski, E. (2008). Blood Gas Analysis and Interpretation. Denver, Colorado: Colorado Association for Continuing Medical Laboratory Education, Inc.

Jarreau, P. (2005). Clinical Laboratory Science Review (3rd ed.). New Orleans, LA: LSU Health Science Center.

Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

16

ELECTROLYTES

Electrolytes Substances whose molecules dissociate into ions

when they are placed in water. Osmotically active particles Classification of ions: by charge

CATIONS (+) In an electrical field, move toward the cathode Sodium (Na), Potassium (K), Calcium(Ca), Magnesium(Mg)

ANIONS (-) In an electrical field, move toward the anode Chloride(Cl), Bicarbonate, PO4, Sulfate

2

ELECTROLYTES

General dietary requirements Most need to be consumed only in small

amounts as utilized Excessive intake leads to increased excretion via

kidneys Excessive loss may result in need for corrective

therapy loss due to vomiting / diarrhea; therapy required - IV

replacement, Pedilyte, etc.

3

ELECTROLYTE FUNCTIONS

Volume and osmotic regulation Myocardial rhythm and contractility Cofactors in enzyme activation Regulation of ATPase ion pumps Acid-base balance Blood coagulation Neuromuscular excitability Production of ATP from glucose

4

ELECTROLYTE PANEL

Panel consists of: sodium (Na) potassium (K) chloride (Cl) bicarbonate CO2 (in its ion form = HCO3

- )

5

ANALYTES OF THE ELECTROLYTE PANEL

Sodium (Na)– the major cation of extracellular fluid Most abundant (90 %) extracellular cation Diet

Easily absorbed from many foods

6

FUNCTION: SODIUM

Influence on regulation of body water Osmotic activity

Sodium determines osmotic activity Main contributor to plasma osmolality

Neuromuscular excitability extremes in concentration can result in neuromuscular

symptoms Na-K ATP-ase Pump

pumps Na out and K into cells Without this active transport pump, the cells would fill with

Na+ and subsequent osmotic pressure would rupture the cells

7

REGULATION OF SODIUM Concentration depends on:

intake of water in response to thirst excretion of water due to blood volume or osmolality

changes Renal regulation of sodium

Kidneys can conserve or excrete Na+ depending on ECF and blood volume by aldosterone and the renin-angiotensin system

this system will stimulate the adrenal cortex to secrete aldosterone.

8

REFERENCE RANGES: SODIUM

Serum 136-145 mEq/L or mmol/L

Urine (24 hour collection) 40-220 mEq/L

9

SODIUM

Urine testing & calculation: Because levels are often increased, a dilution of the

urine specimen is usually required. Once a number is obtained, it is multiplied by the

dilution factor and reported as (mEq/L or mmol/L) in 24 hr.

10

DISORDERS OF SODIUM HOMEOSTASIS Hyponatremia: < 136 mmol/L

Causes of: Increased Na+ loss Increased water retention Water imbalance

Hypernatremia:> 150 mmol/L Causes of:

Excess water loss Increased intake/retention Decreased water intake

11

HYPONATREMIA

1. Increased Na+ loss Aldosterone deficiency hypoadrenalism

Diabetes mellitus In acidosis of diabetes, Na is excreted with

ketones Potassium depletion K normally excreted , if none, then Na

Loss of gastric contents

12

HYPONATREMIA

2. Increased water retention Dilution of plasma Na+ Renal failure Nephrotic syndrome Hepatic cirrhosis Congestive heart failure

13

HYPONATREMIA

3. Water imbalance Excess water intake Chronic condition

14

SODIUM

Note: Increased lipids or proteins may cause false

decrease in results. This would be classified as artifactual/pseudo-hyponatremia

15

CLINICAL SYMPTOMS OF HYPONATREMIA

Depends on the serum level Can affect

GI tract Neurological

Nausea, vomiting, headache, seizures,coma

16

HYPERNATREMIA

1. Excess water loss Sweating Diarrhea Burns Diabetes insipidus

17

HYPERNATREMIA 2. Increased intake/retention

• Excessive IV therapy

3. Decreased water intake • Elderly • Infants • Mental impairment

18

CLINICAL SYMPTOMS OF HYPERNATREMIA

Involve the CNS Altered mental status Lethargy Irritability Vomiting Nausea

19

SPECIMEN COLLECTION: SODIUM

Serum (sl hemolysis is OK, but not gross) Heparinized plasma Timed and random urine Sweat GI fluids Liquid feces (would be only time of excessive

loss)

20

ANALYTES OF THE ELECTROLYTE PANEL

Potassium (K+) the major cation of intracellular fluid

Only 2 % of potassium is in the plasma Potassium concentration inside cells is 20 X greater than it is

outside. This is maintained by the Na-K pump

exchanges 3 Na for 1 K Diet

easily consumed by food products such as bananas

21

FUNCTION: POTASSIUM Critically important to the functions of

neuromuscular cells Acid-base balance Intracellular fluid volume Controls heart muscle contraction Promotes muscular excitability Decreased potassium decreases excitability (paralysis and arrhythmias)

22

REGULATION OF POTASSIUM

Kidneys Responsible for regulation. Potassium is readily

excreted, but gets reabsorbed in the proximal tubule - under the control of ALDOSTERONE

Diet Cell Uptake/Exchange

23

REFERENCE RANGES: POTASSIUM Serum (adults)

3.5 - 5.1 mEq/L or mmol/L Newborns

3.7 - 5.9 mEq/L Urine (24 hour collection)

25 - 125 mEq/L

24

DISORDERS OF POTASSIUM HOMEOSTASIS Hypokalemia

< 3.5 mmol/L Causes of:

Non-renal loss Renal Loss Cellular Shift Decreased intake

Hyperkalemia >5.1 mmol/L Causes of

Decreased renal excretion Cellular shift Increased intake Artifactual/False elevations

25

HYPOKALEMIA 1. Non-renal loss

Excessive fluid loss ( diarrhea, vomiting, diuretics )

Increased Aldosterone promote Na reabsorption … K is excreted in its place

26

HYPOKALEMIA

2. Renal Loss Nephritis, renal tubular acidosis,

hyperaldosteronism, Cushing’s Syndrome

3. Cellular Shift Alkalosis, insulin overdose

4. Decreased intake

27

MECHANISM OF HYPOKALEMIA Increased plasma pH ( decreased Hydrogen ion )

28

K+ moves into RBCs to preserve electrical balance, causing plasma potassium to decrease. ( Sodium also shows a slight decrease )

H+

K+

RBC

CLINICAL SYMPTOMS OF HYPOKALEMIA

Neuromuscular weakness Cardiac arrhythmia Constipation

29

HYPERKALEMIA 1. Decreased renal excretion

Renal disease Addison’s disease Hypoaldosteronism

2. Cellular Shift Such as acidosis, chemotherapy, leukemia,

muscle/cellular injury Hydrogen ions compete with potassium to get into

the cells

30

HYPERKALEMIA

3. Increased intake Insulin IVs promote rapid cellular potassium uptake

4. Artifactual • Sample hemolysis • Prolonged tourniquet use • Excessive fist clenching

31

CLINICAL SYMPTOMS OF HYPERKALEMIA

Muscle weakness Tingling Numbness Mental confusion Cardiac arrhythmias Cardiac arrest

32

SPECIMEN COLLECTION:POTASSIUM

Non-hemolyzed serum heparinized plasma 24 hr urine

33

ANALYTES OF THE ELECTROLYTE PANEL Chloride (Cl-)

the major anion of extracellular fluid

Chloride moves passively with Na+ or against HCO3

- to maintain neutral electrical charge

Chloride usually follows Na if one is abnormal, so is the other

34

FUNCTION: CHLORIDE

Body hydration/water balance Osmotic pressure Electrical neutrality

35

REGULATION OF CHLORIDE

Regulation via diet and kidneys In the kidney, Cl is reabsorbed in the renal proximal

tubules, along with sodium. Deficiencies of either one limits the reabsorption of the

other.

36

REFERENCE RANGES: CHLORIDE Serum

98 -107 mEq/L or mmol/L

24 hour urine 110-250 mEq/L varies with intake

CSF

120 - 132 mEq/L Often CSF Cl is decreased when CSF protein is increased,

as often occurs in bacterial meningitis.

37

DETERMINATION: CHLORIDE

Specimen type Serum Plasma 24 hour urine CSF Sweat

Sweat Chloride Test Used to identify cystic fibrosis patients

Increased salt concentration in sweat Pilocarpine= chemical used to stimulate sweat production Iontophoresis= mild electrical current that stimulates sweat

production

DISORDERS OF CHLORIDE HOMEOSTASIS Hypochloremia

Decreased blood chloride Causes of :

Conditions where output exceeds input

Hyperchloremia Increased blood chloride Causes of:

Conditions where input exceeds output

39

HYPOCHLOREMIA Decreased serum Cl

loss of gastric HCl salt loosing renal diseases metabolic alkalosis/compensated respiratory acidosis

increased HCO3- & decreased Cl-

40

HYPERCHLOREMIA Increased serum Cl

dehydration (relative increase) excessive intake (IV) congestive heart failure renal tubular disease metabolic acidosis

decreased HCO3- & increased Cl-

41

SPECIMEN COLLECTION: CHLORIDE

Serum Heparinized plasma 24 hr urine Sweat

42

ANALYTES OF THE ELECTROLYTE PANEL

Carbon dioxide/bicarbonate (HCO3-)

the major anion of intracellular fluid 2nd most abundant anion of extracellular fluid Total plasma CO2= HCO3

- + H2CO3- + CO2

HCO3

- (bicarbonate ion) accounts for 90% of total plasma CO2

H2CO3

- (carbonic acid)

43

FUNCTION: BICARBONATE ION

CO2 is a waste product continuously produced as a result of cell metabolism, the ability of the bicarbonate ion to accept a hydrogen

ion makes it an efficient and effective means of buffering body pH

dominant buffering system of plasma

44

REGULATION OF BICARBONATE ION

Bicarbonate is regulated by secretion / reabsorption of the renal tubules

Acidosis : ↓ renal excretion Alkalosis : ↑ renal excretion

45

REGULATION OF BICARBONATE ION

Kidney regulation requires the enzyme carbonic anhydrase - which is present in renal tubular cells & RBCs

carbonic anhydrase

Reaction: CO2 + H2O ⇋ H2CO3 → H+ + HCO–3

46

Pulmonary Control Renal Control

REFERENCE RANGE: BICARBONATE ION Total Carbon dioxide (venous)

23-29 mEq/L or mmol/L includes bicarb, dissolved & undissociated H2CO3 - carbonic

acid (bicarbonate)

Bicarbonate ion (HCO3–) 22-26 mEq/L or mmol/L

47

SPECIMEN COLLECTION: BICARBONATE ION

heparinized plasma arterial whole blood fresh serum Anaerobic collection preferred

48

ELECTROLYTE BALANCE

Anion gap – an estimate of the unmeasured anion concentrations such as sulfate, phosphate, and various organic acids.

49

ELECTROLYTE SUMMARY

50

cations (+) Na 142 K 5 Ca 5 Mg 2 154 mEq/L

anions (-) Cl 105 HCO3- 24 HPO4- 22 SO4-2 1 organic acids 6 proteins 16

154 mEq/L

ANION GAP Anion Gap Calculations

1. Na - (Cl + CO2 or HCO3-)

Reference range: 7-16 mEq/L

Or

2. (Na + K) - (Cl + CO2 or HCO3-)

Reference range: 10-20 mEq/L 51

FUNCTIONS OF THE ANION GAP Causes in normal patients

what causes the anion gap?

2/3 plasma proteins & 1/3 phosphate& sulfate ions, along with organic acids

Increased AG – uncontrolled diabetes (due to lactic & keto acids) severe renal disorders Hypernatremia lab error

Decreased AG -

a decrease AG is rare, more often it occurs when one

test/instrument error

52

REFERENCES Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry:

Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

http://thejunction.net/2009/04/11/the-how-to-authority-for-donating-blood-plasma/

http://www.nlm.nih.gov/medlineplus/ency/article/002350.htm Sunheimer, R., & Graves, L. (2010). Clinical Laboratory

Chemistry. Upper Saddle River: Pearson .

53

Water Balance

• Water – 60% of total body mass

– Main Compartments

• Intracellular (ICF) – inside cells – 2/3

• Extracellular (ECF)

– outside cells – 1/3

2

Water Balance

3 Extracellular compartment

More on the ECF…

• Extracellular Compartment(ECF) – Composed of two sub-

compartments • Interstitial fluid (ISF)

– Surrounds cells in tissue • Intravascular fluid (IVF)

– Volume of measurable fluid

– plasma

4

Body Fluid Composition • Plasma

– 55% of total blood volume

– Analytes measured directly

– Consists of ions, molecules, proteins

• Serum

5

Water Balance

• Ions exist in all of these fluids, but the concentration varies depending on individual ion and compartment

• The body uses active and passive(diffusion) transport principles to keep water and ion concentration in place

6

Water Balance

• Plasma proteins – ALBUMIN – Draw water INTO the vessels

• Hydrostatic pressure – Drives water OUT of the vessels

• These two forces create OSMOTIC or ONCOTIC PRESSURE

7

Water balance

• Sodium has a pulling effect on water – More Na outside cells than inside, the water is pulled out of cells

into the extracellular fluid. – Na+ determines osmotic pressure of extracellular fluid

• Proteins (especially albumin) inside the capillaries strongly pulls/keeps

water inside the vascular system – Albumin provides oncotic pressure. – By keeping Na+ & albumin in their place, the body is able to regulate

its hydration.

• When there is a disturbance in osmolality, – the body responds by regulating water intake and urinary control of

water loss or retention, not by changing electrolyte balance

8

Water Balance & Osmolality

Osmolality - • Physical property of a solution based

on solute concentration – Water concentration is regulated by

thirst and urine output – Thirst and urine production are

regulated by plasma osmolality

9

Water Balance & Osmolality

• Increased osmolality stimulates two responses that regulate water – Hypothalamus stimulates the sensation of

thirst – Posterior pituitary secrets arginine

vasopressin hormone (AVP) • AVP increases H2O re-absorption by renal

collection ducts

• In both cases, plasma water increases

10

Osmolality

• Osmolality – concentration of solute / kg – reported as mOsm / kg

• another term:

– Osmolarity - mOsm / L - not often used

11

Osmolality

• Calculated osmolality – uses glucose, BUN, & Na values – Formula:

• 2 (Na) + glucose∕18 + BUN∕2.8 = calculated osmolality

• Osmolal gap – Difference between calculated and determined osmolality

– Formula:

• Determined Osm/kg-calculated Osm/ kg= osmolal gap

• Should be less than 10-15 units difference • (

12

Formulas in Action • A 40-year-old woman suffers from vomiting and

diarrhea. What would be her osmolality based on the below data? – Sodium= 145 mmol/L – Glucose= 750 mg/ dL – BUN= 25 mg/dL

13

Regulation of Blood Volume

• Renin-angiotension-aldosterone system – Aldosterone stimulates sodium

reabsorption and potassium ion secretion

• Natriuretic peptides • Glomerular filtration rate • Volume receptors

14

Renin-Angiotensin-Aldosterone System

• Series of events – Body detects decreased

blood volume – Renin converts

angiotensinogen to angiotension I

– Angiotension I converted to angiotension II by ACE

– Angiotension II causes • Vasoconstriction • Secretion of

aldosterone • Stimulates AVP

secretion and thirst • Enhances NaCl

reabsorption 15

References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry:

Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

• http://thejunction.net/2009/04/11/the-how-to-authority-for-donating-blood-plasma/

• http://www.nlm.nih.gov/medlineplus/ency/article/002350.htm

• Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

16

Overview of Iron

• Essential mineral to most living organisms • Most abundant trace element • 2-2.5 of the 3-5 grams of iron in our bodies is

found in hemoglobin (RBCs and RBC precursors)

Where does iron come from? • Dietary sources - meats,

especially organ meats, spinach, beats,... etc.

Regulation

• Dietary sources • Absorption

– Must be in ferrous state (Fe++) – Occurs in the stomach/small intestines

• Iron “stores” – Iron is recycled when RBCs are broken down – 25% stored in liver, spleen and bone marrow as

ferritin or (Fe3+)

Functions of Iron

• Essential element of heme and hemoglobin • Component of methemoglobin, myoglobin

and some enzymes • Cellular oxidative mechanisms

Heme Sythesis Review

The addition of ferrous iron (Fe++)forms heme

Forms of Iron • Ferrous(Fe2+)

– Absorbed form • Ferric (Fe3+)

– Ferritin – Transport and storage form – Free ferric form is picked up in the plasma by protein transferrin – Delivered to cells having receptor sites

• Gut mucosal cells • Liver cells • RE system cells • Once inside the cell, ferric iron attaches to protein apoferritin to form ferritin • Deficiency of apoferritin results in ferric iron deposits or hemosiderin, which is

insoluble

8

Iron Links

http://www.umm.edu/blood/aneiron.htm http://www.ehendrick.org/healthy/000772.htm http://www.nlm.nih.gov/medlineplus/ency/article/000584.htm http://www.healthservices.gov.bc.ca/msp/protoguides/gps/ferritin.html

Hemoglobin

• Structure, Synthesis, Degradation and Role – Refer to Hematology notes for review

• Chapter 6 in McKenzie text

Porphyrins

• General structure – Cyclic compounds called

tetrapyrroles – Linked by four pyrrole

rings bonded by methene bridges

Porphyrins

• Chemical intermediates in the synthesis of hemoglobin, myoglobin and other respiratory pigments (cytochromes)

• Clinical significance – Presence indicates abnormal heme synthesis

Physical properties

• Color – Coloration around 405 nm – Usually red

• Fluorescence – around 620 nm – Reddish-pink color

• Chelation – Arrangement of nitrogen atoms allows chelation of metal

atoms such as iron, that participate in oxidative metabolism

Porphyrin Synthesis & Control • Synthesis

– Bone marrow and liver are the main site – Some steps of synthesis occur in mitochrondria and cytoplasm of cell

• Control – Enzyme: δ-aminolevulinic acid (ALA)

• Found in liver – Increases in hepatic heme decrease the production of ALA – Decreases or depletions of heme result in ALA increased production – Rate of heme syntheis is flexible and can change rapidily in response

to external stimuli

Porphyrins: Ones to keep an Eye on • Uroporphyrin: URO

– Water soluble – Heme precursor – Found in urine

• Coproporphyrin: COPRO – Water soluble – Heme precursor – Found in urine and feces

• Protoporphyrin: PROTO – Water insoluble – Heme precursor – Found in feces

Porphyrinogens

• Reduced form of porphyrins • Functional precursor of heme • Difficult to measure due to instability and

colorlessness

Glycated hemoglobin

• Hemoglobin A 1c most stable • Indicator of long-term glucose control

– Why? • Reflects sustained average plasma glucose over the RBC

life span

– Correlates with risk of cardiovascular disease and other vascular disorders

Myoglobin

• Heme protein found in skeletal and cardiac muscle • Unable to release oxygen, except under low oxygen

tension • Main function is to transport oxygen from the muscle

cell membrane to the mitochondria • Serves as an extra reserve of oxygen to help

exercising muscle maintain activity longer • Used to diagnose acute myocardial infarction

Lead

• Found in the environment and in paint • Considered a toxin, plays no known role in NORMAL human

physiology • Exposure primarily respiratory or gastrointestinal

• Half-life in whole blood= 2-3 weeks

– Half-life= the time required by the body, tissue or organ to metabolize or inactivate half the amount of substance taken in

Lead • Absorption

– Depends on age, nutritional status and other substances that are present

• Transport – Once in the blood, 94% transferred to RBC bound to hgb – Once it reaches its half-life, lead is distributed to soft tissues, such as

kidneys, liver and brain. Final storage is in soft tissue(5%) and bone (95%)

• Excretion – Urine (76%) – Feces (16%) – Other (8%)

Specimen Requirements: Iron Studies – Serum without anticoagulant – Plasma with heparin ( check product

insert) –Oxalate, citrate or EDTA binds Fe ions, so

they are unacceptable –Early morning sample preferred due to

diurnal variation – No hemolysis

Iron Study/Profiles

% Fe SaturationTotal Iron

TIBCx=

100

3

Three Components ◦ Total Iron ( serum ) ◦ TIBC ◦ % Iron Saturation ( Fe Sat )

The Iron Saturation is a measurement of how “full”

transferrin is

Assessing Iron Levels and Forms Directly measured ◦ Iron ◦ Transferrin Beta globulin formed in the liver Measured by the amount of iron it can bind ◦ Ferritin Best diagnostic test for IDA Acute phase reactant

Assessing Iron Levels and Forms • Indirect measure

– TIBC (Total iron-binding capacity) • Measures the total amount of iron that

apotransferrin can bind • Can be expressed as a percentage(percent

saturation) • Ratio of serum iron to TIBC

• Increased – Late pregnancy – IDA – Following hemorrhage – Following destruction of liver cells

• Decreased – Decreased synthesis of transferrin – Increased loss of urine proteins

Test Methodologies: Iron • Colorimetric Procedure

–Separate Fe from transferrin with a strong acid

–Iron is reduced from ferrous(Fe3+) to ferric(Fe2+) state

–Addition of a chromogen creates a colored compound

–Measurement of colored product by spectrophotometry

Iron Reference Ranges – Diurnal variation

– Men: 65-165 µg/dL

– Women: 45-160

µg/dL

– Decreased Levels • Decreased intake • Increased need • Increased loss

– Increased Levels • Increased absorption • Hemolytic anemia • Lead poisoning • Pernicious anemia • Megaloblastic anemia • Hepatitis

Test Methodologies:TIBC Pre-treatment and Colorimetric Method 1. Add Fe3+ to saturate binding sites on

transferrin 2. MgCO3 is added to remove

unbound Fe3+ 3. Mixture is centrifuged and the

supernatant tested using the serum iron methodology

Reference Ranges Transferrin ◦ 200-360 mg/dL

Ferritin ◦ Male: 20-250 ng/mL ◦ Female: 10-120 ng/mL

TIBC ◦ 250-425 µg/dL

% saturation ◦ 15-55

Test Methodology: Hemoglobin Electrophoresis ◦ Discussed in separate unit

Test Methodology: Porphyrins Screening tests ◦ Urinary PBG ◦ Urinary ALA ◦ Urinayr porphyrins

Quantitative Assays ◦ URO ◦ PROTO ◦ COPRO

Serve to classify porphyrias

Lab Methods Watson-Schwartz for Urinary PBG(

porphobilinogen) ◦ Screen for acute intermittent porphyria ◦ Specimen Qualitative: fresh morning urine Quantitative: 24 hour collection ◦ Reference Range <2 mg/daily

Watson-Schwartz Principle ◦ PBG + Ehrlich’s

reagent results in a red-orange chromogen ◦ Interferences Urobilinogen indole

Lab Methods: HgbA1c Electrophoresis Enzymatic Assays HPLC ◦ Goal is to separate hemoglobin forms within

a column. Then, glycated versus total hemoglobin can be measured spectrophotometrically ◦ Specimen requirements EDTA whole blood Can be non-fasting

Reference range ◦ 4.0-6.0%

Lab Methods: Myoglobin Procedures incorporate the binding of

specific antibodies to myoglobin with a resulting chemical or physical change that can be measured and correlated to myoglobin concentration

Specimen requirements ◦ Usually plasma ( check product insert)

Specimen Requirements: Lead Whole blood ◦ Why? Circulating lead found in the RBC ◦ Venous sample preferred but capillary

sample can be used ( must confirm positive on capillary) ◦ Royal blue top with EDTA anticoagulant ◦ Lead-free containers

Urine

Lab Method: Lead Test methodologies ◦ AAS ◦ Anodic stripping voltammetry

Reference Ranges in blood ◦ Children< 10 µg/dL

Iron Deficiency

• Lab Features – Microcytic, hypochromic anemia – Anisocytosis, poikilocytosis – Total iron and Percent saturation decreased – TIBC increased

Hemosiderosis

• Excessive levels of iron in storage

Hemochromatosis

• Characterized by an increased rate of absorption and less ferritin production

• Excessive iron deposits in organs • Patient develops bronze color in the tissues • Total iron, percent saturation increased • TIBC decreased

Iron Status in Disease States Condition Serum Iron Transferrin Ferritin % Saturation IDA Decreased Increased Decreased Decreased

Iron Overdose Increased Decreased Increased Increased

Hematochromatosis Increased Slight Decrease Increased Increased

Malnutrition Decreased Decreased Decreased Variable

Chronic anemia Decreased Normal/decreased

Normal/increased

decreased

Acute liver disease Increased Variable Increased Increased

Case Scenario #1

• A 40-year-old female is scheduled to have an elective surgery. Her physician ordered a routine CBC pre-op. The following test results were obtained: Test Result Reference Range

Hgb (g/dL) 10 12-16.0

Hct (%) 29.9 42-52

MCV (fL) 75 80-100

MCHC (g/dL) 30 32-36

WBC (x 103/L) 6.0 4.5-11

Plts (x 109/L) 200 150-450

Case Scenario #1

• On review of her blood smear, the technician noted target cells.

• What other types of morphology would we expect to see on this patient?

• The physician then ordered a serum iron, ferritin and TIBC level.

Case Scenario #1

• Below are the results on the additional tests: • What is her diagnosis?

Test Result Reference Range

Serum iron ( µg/dL) 20 65-165

Ferritin ( µg/dL) 5 20-200

TIBC ( µg/dL) 550 260-440

Hemoglobin Disorders

• Refer to Hematology notes – Chapter 10: Hemoglobinopathies – Chapter 11: Thalassemia

Porphyrin Disorders= Porphyrias

• Inherited or Acquired • Enzyme deficiencies resulting in

overproduction of heme precursors in bone marrow or liver

Porphrias

• Classification – Based on

• Specific enzyme deficiency • Hepatic vs erythropoietic • Cutaneous vs neurologic

Porphyrias

• Clinical symptoms – Cutaneous photosensitivity – Itchy skin – Hyperpigmentation – Inflammatory reaction occurs on exposure to

ultraviolet light – Neurologic abnormalities due to increased ALA

and PBG

Porphyrin Conditions • Secondary Conditions

– Porphyrinuria • Increase in coproporphyrin production • Causes

– Lead intoxication – Liver damage – Infection – Accelerated erythropoiesis

– Porphyrinemia • Increase in erythrocytic protoporphyrin concentration • Causes

– Lead intoxication – Iron deficiency – Impaired Iron absorption – Chronic infection

Myoglobin

• Elevations – Acute myocardial – Renal failure – Vigorous exercise – Electric shock – Intramuscular injections

LEAD

• Clinical Features – Children

• CNS symptoms: headache ,clumsiness, seizures, behavioral changes

• GI symptoms: Abdominal pain, colic, constipation

– Adults • Peripheral neuropathies, motor weakness, anemia

Case Scenario #2

• A mother brings her active 2-year-old son to the pediatrician for a routine visit. The physician orders a CBC. Below are the results:

Test Result Reference Range

Hgb (g/dL) 10.2 14-17.4

Hct (%) 30.6 36-46

Case Scenario #2

• The mother reports that her son has had some constipation and abdominal pain. The child does eat well, and the mother gives the child a vitamin supplement, which includes iron

• The mother did mention that they live in an older home that is in need of repainting.

• The physician orders further testing…

Case Scenario #2

• Results of testing

Test Result Reference Range

Serum iron 120 65-165

Ferritin 150 20-200

Whole blood lead (µg/dL)

60 < 10

Erythrocyte protoporphyrin (µg/dL)

150 17-77

What is the diagnosis?

• Lead Poisoning • How does this occur? • Lead inhibits certain

enzymes in the heme synthesis pathway

Case Scenario #2

• IDA was ruled out based on the serum iron and ferritin levels

Functions of the Heart

• Pumps blood to the organs of the body • Delivers oxygen and nutrients where they are

needed • Removes waste products from tissues

Symptoms of Heart Disease

Pathologic Conditions of the Heart

• Congenital Cardiovascular Defects – Abnormality arises from abnormal formation of

heart or its major blood vessels – Present at birth

• All defects develop before the 10th week of pregnancy

– Origin unknown but appear to be based on genetic disposition and environmental influences

Congenital Cardiovascular Defects

• Symptoms – Cyanosis – Pulmonary hypertension – Embolism – Clubbed fingers – Reduced growth – Syncope

• Examples – Tetralogy of Fallot – Ventricular septal defects “hole in the heart”

Pathologic Conditions of the Heart

• Heart Failure or Congestive Heart Failure – Any structural or functional cardiac disorder that impairs

the ability of the ventricle to fill with or eject blood – Result

• Excess fluid accumulates in the lungs producing edema • Reduced output of blood to systemic circulation • Retention of fluid by the kidneys

Heart Failure or Congestive Heart Failure

• Examples – Left ventricular dysfunction – Coronary artery disease – Cardiac arrhythmias

• See it: http://www.youtube.com/watch?v=3cW8__wFXDA

Pathologic Conditions of the Heart

• Acute Coronary Syndromes – Term used to describe a series of events

• Angina • Reversible tissue injury • Unstable angina • Myocardial infarction • Extensive tissue necrosis

Acute Coronary Syndromes

• Clinical Symptoms – Chest pain – Referred pain – Nausea – Vomiting – Dyspnea – Diaphoresis – Light headedness

Acute Coronary Syndromes

• Causes – Atherosclerosis

• Inflammatory disorder • Plaques deposit in

artery walls • Leads to ischemia

Stages of Atherosclerosis 1. Initial vascular injury caused by:

1. Hypertension, hyperlipidemia, hyperhomocysteinemia 2. Increased permeability to lipids especially LDL/VLDL

1. Results in inflammation 3. Monocytes & Leukocytes arrive to help! 4. Macrophages scavenge LDL/cholesterol-rich lipoproteins- become foam cells 5. Foam cells promote lesion progression 6. T and B lymphocytes are recruited by the plaque 7. Interactions between T/B lymphs and foam cells recruits smooth muscle cells into

the lumen 8. Smooth muscle cells secrete collagen, elastin, and proteoglycans to fix the plaque

to the vessel wall • See the process

Presentation of Coronary Heart Disease

Hypertension

• Persistent systolic BP of at least 140 mm HG and/or diastolic BP of at least 90 mm Hg

• Prevalence increases with age • Contributing factors

– Obesity – Physical inactivity – Unhealthy nutrition

Hypertensive Heart Disease

• Term used to describe heart disease caused by direct or indirect effects of increased BP

• Peripheral resistance determining factor in BP – Increases workload of left ventricle resulting in

hypertrophy and dilation of mitral valve. This valve is affected and blood is regurgitated to the left atrium

Infective Heart Disease

• Heart disease caused by infectious agents • Examples

– Rheumatic Heart Disease • Complication of rheumatic fever due to autoimmune response. • Causative organism is Group A streptococcus • Usually affects young adults and children

– Infective Endocarditits • Infection of endocardial surface of the heart • Causative organism Group D streptococcus, but others also

– Pericarditis • Inflammation of the pericardium • Causative agents include bacteria, fungi, viral, autoimmune, others

Diagnosing Heart Disease

• Myocardial Infarction – Diagnosis based on clinical symptoms, EKG

changes and the rise/fall of biochemical markers – Samples collected at onset, 6-9 hours and 12-24

hours if previous samples were negative – Preferred biomarkers are Troponin I and T.

• Specific and sensitive for myocardial necrosis • Current guidelines suggest the use of 2 markers for

diagnosis

Current Cardiac Panel

– Myoglobin • Released from damaged cardiac/skeletal muscle

– Cardiac troponins • See upcoming slide

– CK – CK-MB – BNP

• Discussed later

Time Course Of Enzyme Activity in MI’s

• Historically CK, CK-MB, AST, LD/LDH isoenzymes used

Enzyme Onset of Elevation

Peak activity (Hr)

Duration of Elevation

CK 4-8 12-24 3-4

CK-MB 4-6 12-24 2-3

AST 8-12 24 5

LD 12-24 72 10

LDH isoenzymes

12-24 5

Time Course Of Enzyme Activity in MI’s

• Troponin – Rises 4-10 hours after

onset – Peak at 12-48 hours – Elevated for 4-10 days

• Myoglobin – Released 1-4 hours after

onset • CK-MB

– Rises within 4-6 hours after onset

– Peaks at 12-24 hours – Normal at 2-3 days

Troponin

• Consists of three proteins that bind to thin filament(actin) of cardiac and skeletal muscle – Troponin T (TnT) – Troponin I (TnI) – Troponin C (TnC)

• Function to bind Ca+ and regulate muscle contraction • Absent in the serum of healthy people

What’s So Special About Troponin?

• Specific for cardiac tissue • High diagnostic specificity and sensitivity • Early detection following MI • Remain elevated for several days • Undetected in healthy people • Few interfering substances in detection

Markers of Inflammation

• High Sensitivity C-Reactive Protein (hsCRP) – Acute phase protein – Produced in the liver in response to injury,

infection and inflammation – Increases in CRP correlate with the risk of

coronary artery disease

Markers of Congestive Heart Failure

• Natriuretic peptide – Hormones that include atrial natriuretic peptide

(ANP), B-type natriuretic peptide(BNP), C-type and D-type

– Assist in regulation of cardiovascular homeostasis

– BNP • Released on ventricular stretch or stress to myocytes in the

absence of necrosis • Increased BNP indicates expanded fluid volume such as that

seen in renal failure and CHF

Vascular Inflammation

Plaque Destabilization

Plaque Rupture

Acute Phase Reactant (CRP)

Ischemia

Necrosis (Troponin)

Myocardial Dysfunction (BNP, NT-proBNP)

References

• Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

• http://medicinembbs.blogspot.com/2010/12/congestive-heart-failure.html

• http://www.resverlogix.com/product_development/cardiovascular_disease/atherosclerosis.html

• http://www.youtube.com/watch?v=upb37rbS1dE • http://www.youtube.com/watch?v=3cW8__wFXDA • http://smabiology.blogspot.com/2009_04_01_archive.html • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory

Chemistry. Upper Saddle River: Pearson .

Introduction 2

Organisms rely on the oxidation of complex organic compounds to obtain energy

Three general types of compounds provide chemical energy to our cells

Lipids=Fats Amino acids = Proteins Carbohydrates= Sugars, starches

Carbohydrates 3

Major food source & energy supply of body

Primary source of energy for brain, erythrocytes, retinal cells

Depending on individual diet, 50-90% of the body's carbohydrate intake is in the form of

Grains - cereals, bread Starchy vegetables - potatoes Legumes - beans, peanuts other sources = sugar, molasses, lactose from milk, fructose from fruit

Stored primarily as liver and muscle glycogen

Description and Classification of Carbohydrates

Contain C, H and O molecules Contain a C=O (ketone) and an –OH(aldehyde)

functional group

Classification Based on certain properties

The size of the base carbon chain Location of the CO functional group Number of sugar units Stereochemistry of compound

Chemical Properties 5

Some ( not all ) carbs are reducing substances (donate electrons) Chemical reduction of other substances These sugars must contain an aldehyde or ketone group Reducing sugars

Glucose Maltose Lactose Fructose Galactose

Sucrose is not a reducing substance

Carbohydrate Metabolism

Glucose is primary energy source Nervous tissue can not concentrate or store

carbohydrates, so a steady supply of glucose is needed

Once the level of glucose falls below a certain range, normal function is impaired

Carbohydrate Breakdown

Dietary Carbohydrates

• Mouth • Salivary amylase

Dextrins/ Maltose

• Stomach/Intestines • Pancreatic amylase

Monosaccharide

• Absorption into intestinal mucosa • Delivered to liver

Carbohydrate Breakdown

Ultimate Goal Convert glucose to CO2 and water with ATP as a by-product

Possible channels Converted to liver glycogen and stored Metabolized to CO2 and H2O Converted to keto-acids, amino acids, and proteins Converted to fats and stored in adipose tissue

Biochemical Pathways in Carbohydrate Breakdown

Embden-Meyerhoff pathway Converts glucose to pyruvate/lactate Primary energy source for humans

Hexose monophosphate shunt Oxidizes glucose to ribose and CO2 Produces NADPH as an energy source

Glycogenesis Converts glucose to glycogen

Carbohydrate Metabolism 10

Glycolysis – the conversion of glucose and other hexoses into lactate or pyruvate

Breakdown of glucose for energy production

Glycogenesis – the conversion of glucose to glycogen usually in liver & muscle Excess glucose is converted and stored as glycogen High concentrations of glycogen in liver and skeletal muscle Glycogen is a quickly accessible storage form of glucose

Carbohydrate Metabolism 11

Glycogenolysis – the breakdown of glycogen to form glucose Glycogenolysis occurs when plasma glucose is decreased Occurs quickly if additional glucose is needed Controlled by hormones & enzymes

Gluconeogenesis – the formation of glucose from non-carbohydrate

sources, such as amino acids, glycerol & fatty acids into glucose Occurs mainly in the liver

Glucose

Glycogenesis

Glycogenolysis

Glycolysis

Gluconeogenesis

Carbohydrate Metabolism 13

Also related: Lipogenesis – the conversion of carbohydrates to fatty acids

Fat is another energy storage form, but not as quickly accessible as glycogen

Lipolysis – the decomposition of fat

The sum or net of all of these processes determines the level of blood glucose.

Regulation of Plasma Glucose 14

Organs / systems involved in glucose regulation Liver : Glucose Glycogen Glucose

Muscle

Skeletal & heart

Pancreas Synthesizes hormones Insulin and Glucagon, somatostatin

Other Endocrine glands

Anterior pituitary gland ( growth hormone) Adrenal gland (epinephrine and cortisol) Thyroid gland (thyroxine)

Regulation of Plasma Glucose 15

If plasma glucose is decreased : Glycogenolysis

The liver releases glucose into the plasma (quick response) Gluconeogenesis and lipolysis

If plasma glucose is increased :

Glycogenesis Liver stores glucose as glycogen

Lipogenesis Formation of lipids

Hormones that Regulate Glucose

16

Insulin Most important & only one to decrease glucose

level Synthesized in the Beta cells of the Islets of Langerhans

(in the pancreas) Released when plasma glucose is increased

Action / Effects of insulin

Facilitates glucose entry into cells cell membranes need insulin to be present for glucose to enter

Promotes liver glycogenesis glucose to glycogen

Promotes glycolysis speeds up utilization of glucose in cells

Promotes synthesis of lipids from glucose Such as the formation of Triglycerides

Promotes amino acid synthesis from glucose intermediates

Decreases / inhibits glycogenolysis and gluconeogenesis

Insulin Control 18

Insulin secretion controlled by: Blood glucose level Certain Amino Acids ie. leucine, & arginine

Counterregulatory Hormones 19

Glucagon 2nd most important glucose regulatory hormone

Referred to as a hyperglycemic agent

Synthesized in alpha cells of the islets of Langerhans

Action/Effect of Glucagon 20

Stimuli – decreased plasma glucose Action Increases glycogenolysis & gluconeogenesis Promotes breakdown of fatty acids Promotes breakdown of proteins to form amino acids Increases plasma glucose concentration

Other Regulatory Hormones 21

Epinephrine One of two glucose regulating hormones from the adrenal gland

Origin – adrenal medulla Action/effect Inhibits insulin secretion & release Promotes lipolysis Stimulates glycogenolysis Immediate release of glucose

Stimuli Neurogenic - based on physical / emotional stress. Adrenal tumors

Other Regulatory Hormones 22

Glucocorticoids - such as cortisol Origin – adrenal cortex

Effect – antagonistic to insulin

increases blood glucose promotes gluconeogenesis from breakdown of proteins inhibits the entry of glucose into muscle cells

Stimuli – anterior pituitary’s ACTH

Other Regulatory Hormones 23

Growth Hormone (GH) and Adrenocorticotropic Hormone (ACTH) Origin – anterior pituitary gland Effect – antagonistic to insulin

Increases plasma glucose levels inhibits insulin secretion inhibits entry of glucose into muscle cells inhibits glycolysis inhibits formation of triglycerides from glucose

Stimuli decreased glucose stimulates its release increased glucose inhibits its release

Other Regulatory Hormones 24

Thyroid hormones (such as thyroxine) Origin – thyroid gland Effect

increases absorption of glucose from intestines Promotes comversion of liver glycogen to glucose

Stimuli – pituitary gland’s TSH

Other Regulatory Hormones

Somatostatin Origin-Delta cells of the islets of Langerhans in the pancreas Effect - increase plasma glucose Actions

antagonistic to insulin, inhibits endocrine hormones including glucagon & growth

hormone

References

Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

26

Laboratory testing Considerations ◦ Reference values depend on: Type of specimen venous/capillary Serum, plasma, whole blood

How was it collected? fasting, random, after a meal

Reference value (serum/plasma) ◦ 74-106 mg/dL

2

Laboratory testing Glucose preservation

Perform testing < 1 hour after collection Separate plasma from cells < 1 hour Cells continue to utilize glucose at a rate of 10 mg/dL per

hour. Refrigeration slows the process.

Collect blood in sodium fluoride tube Grey top tube Fluoride inhibits glycolysis

3

Specimen Collection Whole blood – ◦ Point of care ◦ Results are @ 11% lower than

plasma/serum Serum Plasma

4

Other Specimen Types CSF specimens ◦ Analyzed ASAP ◦ Glucose level is 60-70% of pts current blood level. ◦ CSF glucose in Fasting (non-diabetic) @ 40-70 mg/dL Decreased CSF glucose values suggest bacterial meningitis

because bacteria are consuming glucose as an energy source Normal or Increased CSF glucose suggests viral meningitis.

24 hour urine ◦ A small amount of glucose is lost in the urine daily. Usually

< 500mg/24 hr. ◦ Random urine for diagnosis no longer performed, but

some patients use it for self monitoring.

5

Methods for Glucose Determination

Glucose Oxidase Methodology

7

Glucose + O2 + H2O

Glucose Oxidase Gluconic acid + H2O2

H2O2 + Chromogen Oxidized chromogen + H2O

Peroxidase

Trindler reaction

Glucose oxidase – an enzyme that will catalyze the reaction of glucose to gluconic acid, with the formation of hydrogen peroxide as a by-product

Glucose oxidase Good methodology, but:

◦ Procedure is good for blood and CSF specimens, but urine

has too many interfering substances.

◦ Subject to interference from ascorbic acid, bilirubin and uric acid which are also oxidized by peroxidase. ◦ Alternative way to determine concentration:

(polarographically) • Measuring the amount of oxygen used up by an

electrode

8

Hexokinase

An enzyme that catalyzes the phosphorylation of glucose ◦ Method can be very accurate and precise since

the coupling reaction is specific ◦ Time consuming for routine use ◦ Reference methodology since it lacks

interferences associated with glucose oxidase method ◦ Procedure can utilize blood, urine and CSF

9

Hexokinase Methodology

10

Glucose + ATP

Hexokinase Glucose – 6 – Phosphate + ADP

Glucose – 6 - Phosphate + NADP

G6PD NADPH + H + 6-Phosphogluconate

NADP - Nicotinamide adenine dinucleotide phosphate (oxidized form) is reduced NADPH - reduced form absorbs light (340nm) proportional to the amount of glucose present in first reaction

Laboratory Diagnosis

Laboratory Tests Fasting blood sugar (FBS) ◦ Most frequently ordered “screening” test

for glucose metabolism Reference value: 74-106 mg/dL Fasting values > 126 mg/dL usually indicate a

problem FBS should be repeated on another day to

confirm diagnosis

Borderline diabetes may have a normal FBS & may need a challenge test to demonstrate abnormality

12

Laboratory Tests

2 Hour Postprandial Patient has FBS drawn Ingests a 75 gram high carbo breakfast –

or sometimes drinks glucola Has repeated glucose test at 2 hours Glucose level should have returned to

fasting levels. If glucose > 200 mg/dL on the

postprandial test, a fasting or random glucose level, should be performed on a subsequent day to diagnose with diabetes

13

Laboratory Tests Oral glucose tolerance test (GTT)

No longer recommended by the new ADA

guidelines Used to screen for gestational diabetes • Problems included calculation dosage, patient

must drink it, keep it down, stay relatively inactive during test period, and be successfully drawn “on time”.

14

Oral glucose tolerance test (GTT)

Patient directions - important. ◦ Eat an adequate carbohydrate diet at least three (3) days

prior to test ◦ Evening before the test, no eating after supper meal ◦ Test is begun in early a.m. ◦ Obtain fasting specimen ◦ Test dose: ** test dose has been reduced to 75 gm for

adults and 1.75 gm / kg for children. Test dose must be consumed within 5 minutes.

◦ Patient is to remain resting, no smoking or eating during test period

◦ Blood and urine specimens are collected at hourly intervals

- Testing of the urine glucose & ketones, no longer routine.

15

Oral glucose tolerance test (GTT)

Response to Oral Glucose Tolerance TestResponse to Oral Glucose Tolerance Test

Normal

Abnormal

Laboratory Tests: Ketones Produced by the liver Metabolism by-products of fatty acids Three bodies ◦ Acetone (2%) ◦ Acetoacetic acid (20%) ◦ 3-β hydroxybutyric acid (78%)

Increase in cases of carbohydrate deprivation

or decreased carbohydrate use (diabetes mellitus, starvation/fasting, prolonged vomiting etc.)

Laboratory Tests: Microalbumin • Microalbumin

• Persistent albuminuria in the range of 30-299 mg/ 24 h or an albumin-creatinine ratio of 30-300 µg/mg

• Indication of renal nephropathy • Assists in the diagnosis of early proteinuria • Normal urine dipsticks are insensitive to low

concentrations of urine albumin

Glycosylated Hemoglobin/ Hemoglobin A1c

Long term glycemic control indicator, reflects average blood glucose level over the previous 2-3 months

Glucose molecule attaches nonenzymatically to the hemoglobin molecule

Advantages: ◦ “Time average glucose” not subject to

temporary variability due to diet and exercise

◦ Does not require fasting

Influenced by: ◦ Conditions that affect the life span of the

RBC, such as sickle cell disease and hemolytic diseases

◦ Hemoglobin A1C is the most commonly measured glycosylated hemoglobin

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Glycosylated Hemoglobin/ Hemoglobin A1c Specimen : EDTA whole blood ◦ doesn’t need to be fasting

Measured by electrophoresis, enzymatic assays, HPLC

Hemoglobin A1C reference range ◦ 4.0 - 6.0 %

For diagnosis of diabetes based on Hemoglobin A1C results, the patient must has a result of > 6.5% , confirmed by repeat measurement.

Other related tests: Lactose Tolerance ◦ Lactose - disaccharide ◦ Lactose malabsorption or lack of enzyme

needed to breakdown lactose ◦ Often results in diarrhea, cramping, and gas

– Lab evaluation

– Perform OGTT using lactose, not glucose ◦ Normal GTT curve similar to OGTT (glucose level will

increase 25 mg/dL above the fasting level). ◦ Lactase deficiency Flat curve - no/very little increase in glucose level.

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Urine Glucose Copper Reduction- Clinitest

Not specific Detects all reducing sugars Used to detect galactosemia in babies

and children < 3 yrs old.

References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry:

Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

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Hyperglycemia Increase in plasma glucose levels due to hormone imbalance Healthy patients – Insulin is secreted by the β

cells of the pancreatic islets of Langerhans

Reference Range – Increased plasma glucose:

• > 110 mg / dl – Glucose reference range:

• 74 - 106 mg / dl

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Effects of Hyperglycemia

Immediate Effects – Increased extracellular osmotic pressure

• The increased glucose in plasma pulls water out of cells

• Results in dehydration

– Acidosis - metabolic acidosis. • May result • If the patient’s cells are not able to take in

glucose, they may begin to convert fats to fatty acids, which then become keto acids.

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Effects of Hyperglycemia: Long term Physiological – Heart attacks/strokes, Diabetic

retinopathy(Blindness), kidney failure, neurologic defects, susceptibility to infections

Chemical – Glycosylated hemoglobin

• the formation of glycosylated hemoglobin is the result of prolonged elevation of plasma glucose.

Diabetes

Characterized by hyperglycemia Disorders differ in etiology, symptoms and consequences Lab’s role – Assist in diagnosis of the disease – Identification of the disorder – Assessment of progression of tissue damage

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Physiologic abnormalities of diabetes

Hyperglycemia – increase blood glucose. – Doesn’t matter how the glucose is derived - diet, fat

metabolism, protein destruction/wasting Ketosis – from fat metabolism, ketonemia, ketonuria

Hyperlipidemia -increase blood lipids from faulty glucose metabolism. Decrease blood pH - metabolic acidosis Urine abnormalities – Glycosuria – glucose present – Polyuria - increase in urine volume – Loss of electrolytes - washing out with the urine

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Diabetes – World Health Organization (WHO) and American

Diabetes Association (ADA) recommends four categories of diabetes:

• Type 1 diabetes – Most severe and potentially lethal

• Type 2 diabetes • Other (secondary diabetes) • Gestational diabetes mellitus (GDM)

Type 1 Diabetes

Insulin dependent diabetes mellitus ( IDDM ) 5-10 % of diabetes cases Demographics – Non-Hispanic Whites/ Non-Hispanic Blacks – Children & adolescents

Pathology – Disease triggered by viral illness or environmental factors that

destroys beta cells in pancreas. – Absolute Insulin deficiency

• Defect in secretion, production or action or all • Autoimmune destruction of islet beta – cells in pancreas • Auto-antibodies are present

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Type 1 Diabetes Clinical Symptoms – CLASSIC TRIAD

• Polyphagia (increased food uptake)

• Polydipsia (thirst) • Polyuria ( increased

urine production)

– Other symptoms • Mental confusion • Rapid weight loss • Hyperventilation • Diabetic ketoacidosis

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Laboratory Findings

Hyperglycemia- plasma levels > 110 mg/dL Glucosuria- plasma glucose > 180 mg / dl

Decreased insulin Increased glucagon – Stimulation causes

• Gluconeogenesis • Lipolysis (breakdown of fat produces ketones)

Ketoacidosis Decreased blood pH ( acidosis ) ↓ Sodium … ↑ Potassium … ↓ CO2

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Type II Diabetes

Non – Insulin Dependent Diabetes Mellitus( NIDDM ) Most common form of diabetes Demographics – Adult onset – Patients usually > 20 years old – American Indians and non-Hispanic blacks

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Type II Diabetes: Pathology Develops gradually Disorder in insulin resistance and relative deficiency of insulin Plasma glucose is unable to enter cells

Contributory factors – Obesity – Lack of exercise – Diet – Genetics – Drugs, such as diuretics, psychoactive drugs – Increases in hormones that inhibit/antagonize

insulin (GH & cortisol)

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Laboratory Findings

Hyperglycemia Glucosuria Insulin is present Glucagon is not elevated

No lipolysis and no ketoacidosis Excess glucose is converted to triglycerides ( ↑ plasma triglycerides )

Normal / Increased Na / K Increased BUN & Creatinine ( Decreased renal function ) Hyperosmolar plasma from hyperglycemia

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Other (SecondaryDiabetes)

Genetic defects of beta cell function Genetic defects in insulin action Genetic syndromes Pancreatic disease Endocrinopathies Drug or chemical induced

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Gestational Diabetes

Glucose intolerance associated with pregnancy’s hormonal and metabolic changes

Mothers usually return to normal after pregnancy, but with increased risk for diabetes later on in life

Infants are at increased risk for respiratory complications and hypoglycemia after birth

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Criteria for Diagnosis of Diabetes

1. Symptoms of diabetes plus random plasma glucose concentration > 200 mg/dL. Random is defined as any time of day without regard to time

OR 2. Fasting plasma glucose > 126 mg/dL. Fasting is defined as no caloric intake

for at least 8 hours. OR 3. 2-Hour postprandial glucose > 200 mg/dL during an oral glucose tolerance

test OR 4. A HgbA1C > 6.5%, confirmed on repeat measurement

Side notes • Glucose tolerance testing ( GTT ) is considered to be of limited additional

use in the diagnosis of diabetes and not recommended, do 2 hour pp test as stated above.

• Urine glucose testing is also not recommended in diabetes diagnosis

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Hypoglycemia Plasma glucose level falls below 60 mg/dL

Glucagon is released when plasma glucose is < 70 mg / dL to inhibit insulin Epinephrine, cortisol, and growth hormone released from adrenal gland to increase glucose metabolism and inhibit insulin

Treatment – Varies with cause. Generally, hypoglycemia is

treated with small, frequent meals, (5-6 / day) low in carbohydrates, high in protein

Hypoglycemia

Symptoms Increased hunger Sweating Nausea Vomiting Dizziness Shaking Blurring of speech and sight Mental confusion

Lab Findings Decreased plasma glucose

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Whipple’s Triad •Symptoms of hypoglycemia •Low plasma glucose at time of symptoms •Alleviation of symptoms with glucose ingestion

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Hypoglycemia Causes of: – Reactive

• Insulin overdose in diabetics • Ethanol ingestion

– Fasting • Insulin-producing tumors • Hepatic dysfunction • Sepsis

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Galactosemia

Resulting from : – Galactose 1, phosphate uridyl transferase deficiency

• enzyme that converts galactose to glucose, patients cannot change either galactose or lactose into glucose.

• results in galactosemia (galactose in blood)

Effects: – Can lead to mental retardation, cataracts, death

check children < 3 yrs for reducing substances

References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. Centers for Disease Control. (2012). Diabetes Public Health Resource. Retrieved from http://www.cdc.gov/diabetes/pubs/factsheet11.htm Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson . http://crossfitovercome.com/2011/12/29/diabetes-primer/

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Terms

Acid Any substance that can yield a hydrogen ion

(H+) or hydronium ion when dissolved in water Release of proton or H+

Base Substance that can yield hydroxyl ions (OH-) Accept protons or H+

Terms

pK/ pKa Negative log of the ionization constant of an acid Strong acids would have a pK <3 Strong base would have a pK >9

pH

Negative log of the hydrogen ion concentration pH= pK + log([base]/[acid]) Represents the hydrogen concentration

Terms

Buffer Combination of a weak acid and /or a

weak base and its salt What does it do?

Resists changes in pH

Effectiveness depends on pK of buffering system pH of environment in which it is placed

Terms Acidosis

pH less than 7.35

Alkalosis pH greater than 7.45

Note: Normal pH is 7.35-7.45

Acid-Base Balance

Function Maintains pH homeostasis Maintenance of H+ concentration

Potential Problems of Acid-Base balance

Increased H+ concentration yields decreased pH Decreased H+ concentration yields increased

pH

Regulation of pH

Direct relation of the production and retention of acids and bases

Systems Respiratory Center and Lungs Kidneys Buffers

Found in all body fluids Weak acids good buffers since they can tilt a reaction

in the other direction Strong acids are poor buffers because they make the

system more acid

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Blood Buffer Systems

Why do we need them? If the acids produced in the body from the

catabolism of food and other cellular processes are not removed or buffered, the body’s pH would drop

Significant drops in pH interferes with cell enzyme systems.

Blood Buffer Systems

Four Major Buffer Systems Protein Buffer systems

Amino acids Hemoglobin Buffer system

Phosphate Buffer system Bicarbonate-carbonic acid Buffer system

Blood Buffer Systems

Protein Buffer System Originates from amino acids

ALBUMIN- primary protein due to high concentration in plasma

Buffer both hydrogen ions and carbon dioxide

Blood Buffering Systems

Hemoglobin Buffer System Roles Binds CO2 Binds and transports hydrogen and

oxygen Participates in the chloride shift Maintains blood pH as hemoglobin

changes from oxyhemoglobin to deoxyhemoglobin

Oxygen Dissociation Curve

Curve B: Normal curve Curve A: Increased affinity for hgb, so oxygen keep close Curve C: Decreased affinity for hgb, so oxygen released to tissues

Bohr Effect

It all about oxygen affinity!

Blood Buffer Systems

• Phosphate Buffer System • Has a major role in the elimination of H+

via the kidney • Assists in the exchange of sodium for

hydrogen • It participates in the following reaction

• HPO-24 + H+ H2PO –

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• Essential within the erythrocytes

Blood Buffer Systems

Bicarbonate/carbonic acid buffer system Function almost instantaneously Cells that are utilizing O2, produce CO2, which

builds up. Thus, more CO2 is found in the tissue cells than in nearby blood cells. This results in a pressure (pCO2).

Diffusion occurs, the CO2 leaves the tissue through the interstitial fluid into the capillary blood

Bicarbonate/Carbonic Acid Buffer

Carbonic acid

Bicarbonate

Conjugate base

Excreted in urine

Excreted by lungs

Bicarbonate/carbonic acid buffer system How is CO2 transported?

5-8% transported in dissolved form A small amount of the CO2 combines directly

with the hemoglobin to form carbaminohemoglobin

92-95% of CO2 will enter the RBC, and under the following reaction CO2 + H20 H+ + HCO3

-

Once bicarbonate formed, exchanged for chloride

Henderson-Hasselbalch Equation

Relationship between pH and the bicarbonate-carbonic acid buffer system in plasma

Allows us to calculate pH

Henderson-Hasselbalch Equation

General Equation pH = pK + log A-

HA Bicarbonate/Carbonic Acid system

o pH= pK + log HCO3

H2CO3 ( PCO2 x 0.0301)

Henderson-Hasselbalch Equation

1. pH= pK+ log H HA 2. The pCO2 and the HCO3 are read or derived from the blood gas analyzer

pCO2= 40 mmHg HCO3

-= 24 mEq/L

3. Convert the pCO2 to make the units the same pCO2= 40 mmHg * 0.03= 1.2 mEq/L

3. Lets determine the pH: 4. Plug in pK of 6.1

5. Put the data in the formula pH = pK + log 24 mEq/L

1.2 mEq/L pH = pK + log 20 pH= pK+ 1.30 pH= 6.1+1.30 pH= 7.40

The Ratio…. Normal is : 20 = Bicarbonate = Kidney = metabolic 1 carbonic acid Lungs respiratory The ratio of HCO3

- (salt/bicarbonate) to H2CO3 (acid/carbonic acid) is normally 20:1

Allows blood pH of 7.40

The pH falls (acidosis) as bicarbonate decreases in relation to carbonic acid

The pH rises (alkalosis) as bicarbonate increases in relation to carbonic acid

Physiologic Buffer Systems

Lungs/respiratory Quickest way to respond, takes minutes

to hours to correct pH by adjusting carbonic acid

Eliminate volatile respiratory acids such as CO2

Doesn’t affect fixed acids like lactic acid Body pH can be adjusted by changing

rate and depth of breathing “blowing off” Provide O2 to cells and remove CO2

Physiologic Buffer Systems

Kidney/Metabolic Can eliminate large amounts of acid Can excrete base as well Can take several hours to days to correct pH Most effective regulator of pH

If kidney fails, pH balance fails

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References

Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

Carreiro-Lewandowski, E. (2008). Blood Gas Analysis and Interpretation. Denver, Colorado: Colorado Association for Continuing Medical Laboratory Education, Inc.

Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

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