Exam 2 + Final Review Content (PDFs)

Clinically Significant Enzymes, Non-Protein Nitrogens [NPN], Liver, Vitamins and Minerals, Tumor Biomarkers, **NO Endocrinology .. 17 pgs**

Enzymes - Influences

• temperature

• pH

• substrate concentration

• coenzymes

• cofactors

Clinically Significant Enzymes

• AST

• ALT

• ALP

• GGT

• LD

• CK

• Amylase

• Lipase

• ACP

AST Purpose/Source

NON-specific → skeletal muscle, cardiac muscle and parenchymal hepatic cells - pyridoxine (Vitamin B6) is a coenzyme of transaminases

AST Test

Kinetics method using aspartate as substrate and converting NADH to NAD causing a decrease in Absorbance

ALT Purpose/Source

sensitive and specific indicator of liver damage, even before hyperbilirubinemia. Hepatocellular damage may elevate ALT levels in serum > 10- 100 times normal levels

ALT Test

• Alanine + a-ketoglutarate <-> (ALT) pyruvate + glutamate

• Pyruvate + NADH + H+ <-> (LD) lactate + NAD+

• Decrease in NADH is measured at absorbance 340 nm

ALP Purpose/Source:

Sensitive indicator of cholestasis & extrahepatic obstruction. Also increases in bone disorders

ALP Test:

p-Nitrophenylphosphate <-> (ALP, pH 10.2) p-Nitrophenol + phosphate ion measures p-nitrophenol

GGT - Purpose/Source:

Found in many tissues but serum levels arise from hepatic biliary damage/chronic alcohol use

GGT - Test

y-glutamyl-p-nitroanilide + glycylglycin ➔ y-glutamyl-glycylglycine ➔ (GGT, pH 8.2) p-Nitroaniline

LD - Purpose/Source:

• LD1>LD2 cardiac disease

• LD 4 & 5 hepatic disease

LD - Test:

Pyruvic acid + NADH ➔ lactate + NAD Immunoassay & electrophoresis

CK - Purpose/Source

• CK MB - cardiac [~5% of total]

• CK BB - brain

• CK MM - skeletal

CK - Test

Creatine + ATP ➔CK ➔ creatine phosphate + ADP + PPP ➔ pyruvate + ATP + NADH ➔ lactate + NAD Immunoassay & electrophoresis

Amylase - Purpose/Source

Pancreas

Amylase - Test

• Starch (substrate) + iodine (indicator) in absence of amylase ➔ loss of blue color

• Kinetic methods employ malto-tetraose as a substrate producing maltose; the method is coupled with enzymatic reactions using NAD ➔ NADH

Lipase: Purpose/Source

• oil substrate: measure loss of turbidity (UV absorption) due to hydrolysis of oil by lipase

• Enzymatic method couples hydrolysis production of fatty acids with color production

ACP: Purpose/Source

Prostate

ACP: Test

p-nitrophenolphosphate <-> (ACP, pH5) p-nitrophenol + phosphate ion

Non-Protein Nitrogens [NPN]

are important for assessing kidney function and nitrogen balance in the body.

• Urea

• Creatinine

• Uric Acid

• Ammonia

Urea

produced in the liver from the breakdown of amino acids; used to assess the function of the kidney

• Pre-renal uremia: dehydration, shock, blood loss, cardiac failure, high protein diet

• Renal uremia: glomerular, tubular and renal vascular dysfunction

• Post-renal uremia: obstruction to renal flow

Renal uremia:

Condition + Urea + Creatinine + BUN:Creatinine

• Normal person with normal diet + 6-20 mg/dL + 0.7-1.3 mg/dL + 12 to 20:1 + Usually 12 to16:1

• Low protein intake Starvation: Severe liver disease + ↓ + V + ↓

• Pre-renal uremia: High protein intake GI hemorrhage ↑ + N + ↑

• Post-renal obstruction: ↑ + ↑ + ↑

Creatinine:

• produced from muscle creatine by CK [so related to muscle mass]; filtered through renal glomerulus but not reabsorbed significantly [so is a good estimate of glomerular filtration function]

  • Estimates glomular function but also increased in muscle wasting diseases

Uric acid:

breakdown product of purines; increased body fluid uric acid may form crystals in joints, with pain and inflammation, in urine, forming calculi

• Gout, kidney stones

Ammonia:

• breakdown of amino acids in liver; normally converted to urea and excreted through kidney; increased in

  • Hepatic coma:

    • Terminal stages of cirrhosis: Reyes Syndrome

Urea Lab Tests:

• Urease method

  • Urea + H2O –> (urease) 2NH4+ + CO -2

  • NH 3+ + pH indicator ➔ color change

  • NH4 ++2-oxoglutarate + NADH ➔ (GLDH) NAD+ + glutamate + H2O

• Monoxime method: Dicetyl monoxime + H2O ➔ (H+) diacetyl + HONH2 Urea + diacetyle ➔ (H+) diazine (yellow) + 2H2O

• Conductimetric

• Berthelot

Creatinine Lab Tests: Jaffe reaction

• Creatine + picrate ➔ (OH-) red-orange complex

• Reacted with alkaline picrate to form colored complex [can be timed to lower interference by non-creatinine chromogens]

Creatinine Lab Tests: Creatininase method

• Creatinine + H2O ➔ (creatininase) creatine

• Creatine + ATP ➔ (CK) creatine phosphate + ADP

• ADP + PEP ➔ (PK) pyruvate + ATP

• Pyruvate + NADH + H+ ➔ (LD) lactate + NAD+

Creatinine Lab Tests: Creatinine clearance

small number means creatinine is not filtered through the glomerulus

• Urine Creat x 24 hour urine volume x 1.73 m² in units of ml per minute Plasma Creat x 1440 min x patient SA in m²

OR

• Urine Creat x 24 hour urine volume. in units of ml per minute Plasma Creat x 1440 min

Uric Acid Lab Tests: Phosphotungstic acid method

Uric acid + H3PW12O40 + O2 ➔ (Na2CO3/OH-) allantoin + tungsten blue + CO2

Uric Acid Lab Tests: Enzyme method

• Uric acid + O2 + 2H2O ➔ (uricase) allantoin + CO2 + H2O2

• Coupled I: H2O2 + CH3OH ➔ (catalse) H2CO + 2H2O

  • CH2O + 3C5H8O2 + NH3 ➔ 3H2O + colored compound

• Coupled II: H2O2 + indicator ➔ (peroxidase) colored compound + 2H2O

Ammonia Lab Tests:

• sensitive to temperature changes [levels increase]; must be placed on ice after collection

• Enzymatic method

  • Glutamate dehydrogenase consumes NADPH with ammonia

  • Decrease in absorbance at 340 nm

• ISE

  • Ammonia diffuses across a semipermeable membrane

  • Change in pH of ammonium chloride solution

Ammonia Ion-Selective Electrode (ISE) Purpose

• Sensor for measuring ammonium ion (NH4+) concentration.

• Operates on potentiometry principle.

• Generates voltage proportional to ammonium ion activity.

• Valuable for assessing metabolic disorders, monitoring kidney function, detecting ammonia toxicity.

• Provides rapid, accurate measurements with minimal sample volume.

Bilirubin Metabolism Diagram

The process by which bilirubin is formed from the breakdown of hemoglobin, processed in the liver, and excreted in bile. It involves conjugation to glucuronic acid, making it water-soluble for elimination from the body through urine

Bilirubin Metabolism Significance

significance includes its role in diagnosing liver diseases and jaundice, as elevated levels indicate liver dysfunction or hemolysis.

Prehepatic (hemolytic) jaundice Diagram

A type of jaundice caused by excessive breakdown of red blood cells, leading to increased bilirubin production before it reaches the liver. This results in elevated unconjugated bilirubin levels in the blood.

Intrahepatic Jaundice Diagram

A type of jaundice that occurs due to liver disease or damage, affecting the liver's ability to conjugate and excrete bilirubin, resulting in elevated conjugated bilirubin levels in the blood.

Posthepatic Jaundice Diagram

A type of jaundice caused by obstruction of bile flow after bilirubin has been processed by the liver, leading to elevated conjugated bilirubin levels in the blood.

Bilirubin Ranges

Normal Range:

• Adult:

  • Conjugated 0-0.2mg/dL

  • Unconjugated 0.2-0.8 1mg/dL

  • Total 0.2-1mg/dL

• Infants: Total Bilirubin 2-6mg/dL

Pre-Hepatic Conditions:

Hemolytic Anemia

• Direct Bilirubin: Increase

• Total Bilirubin: Increase

• Urine Bilirubin: Negative

• Urine Urobilinogen: Increased

• Fecal Urobilinogen: Increased

Hepatic Conditions:

Hepatitis

• Direct Bilirubin: Increase

• Total Bilirubin: Increase

• Urine Bilirubin: Positive

• Urine Urobilinogen: Increased

• Fecal Urobilinogen: Variable

Post-Hepatic Conditions:

Obstruction of bile duct

• Direct Bilirubin: Increased

• Total Bilirubin: Increased

• Urine Bilirubin: Positive

• Urine Urobilinogen: Decreased/Negative

• Fecal Urobilinogen: Decreased/Negative

Types of Jaundice

• Pre-Hepatic

• Hepatic

• Post Hepatic

Prehepatic Jaundice: Unconjugated Hyperbilirubinemias

• Hemolytic Anemia (any cause)

• Ineffective erythropoiesis (ex. Pernicious anemia)

  • Produces more bilirubin than Liver can remove

  • Serum bilirubin levels rarely exceed 5 mg/dL

  • Bilirubin does not appear in the urine

Prehepatic Jaundice

• Neonatal physiological jaundice

  • Enzymes necessary for metabolism and conjugation of bilirubin are not present or newborn with Rh or ABO incompatibility

• Bili >15 mg/dL→ Kernicterus (Unconjugated Bili in CNS causing severe neurologic damage) or Bili >10 mg/dL for more than 2 weeks

• HDN Treatment is phototherapy

Hepatic Jaundice

• Impaired cellular uptake

• Defective conjugation

  • Lack of UDP-glucuronyl transferase (any cause)

• Abnormal secretion of bilirubin from hepatocytes

• Unconjugated Hyperbilirubinemias

• Conjugated Hyperbilirubinemias

Unconjugated Hyperbilirubinemia

high levels of unconjugated (indirect) bilirubin in the bloodstream (decreased hepatic uptake and conjugation)

• Gilbert’s syndrome

• Crigler-Najjar syndrome

Gilbert’s syndrome

• Inherited condition (least serious)

• Impaired cellular uptake of bilirubin

• ↑ bilirubin < 3 mg/dL (unconjugated)

Crigler-Najjar syndrome

• rare, inherited condition

• Absence of enzyme (UDP-glucuronosyltransferase) or decreased enzyme activity

• Patients die within the first year of life due to severe jaundice and kernicterus

Conjugated Hyperbilirubinemias - Dubin-Johnson Syndrome

• An inherited condition

• Lack of transport molecule into bile canal causing defective excretion by liver cells

• Produces an obstructive liver disease that reduces biliary excretion of conjugated bilirubin

Posthepatic Jaundice

Obstructive (Posthepatic) jaundice

• Blockage (mechanical obstruction) of the flow of bile from the liver into the intestine

• Significantly Increased in Conjugated bilirubin in serum, stool appears pale.

Biliary artesia

• Lack of development of bile canal (posthepatic)

• Acquired defect

Other Disorders of Liver

• Cirrhosis

• Tumors

• Reye Syndrome

Cirrhosis

• Scarring of the liver is permanently damaged→ End stage of a number of diseases such as hepatitis

• Caused by alcohol abuse, hemochromatosis (iron toxicity), post necrotic hepatitis,

• primary biliary cirrhosis (an autoimmune disorder) and Hepatitis

Reye Syndrome

A rare condition causing liver and brain swelling, mainly in children post-viral infections like influenza or chickenpox, often linked to aspirin use. Symptoms include vomiting, confusion, and seizures

Liver enzymes used in diagnosis

• Found within Hepatocytes → highly active → high enzyme activity

• Most common enzymes

  • AST (SGOT) -- aspartate aminotransferase

  • ALT (SGPT) -- alanine aminotransferase

  • LD -- lactate dehydrogenase

Common Liver Enzymes Source:

• AST: in heart , liver, muscle and RBC (non-specific)

• ALT (more liver specific) also in heart, kidney, skeletal muscle and rbc

• LD also in muscle, heart and rbcSource of liver enzymes used in diagnosis includes hepatocytes, where they are highly active and released into the bloodstream during liver damage.

Liver Enzyme Changes in Biliary Obstruction and Hepatic Disease

• If bile duct is obstructed or bile canals inflamed, then hepatocytes become inflamed too → ↑ AST, ↑ ALT, ↑ LD

• If true hepatic disease, then ↑↑↑ AST, ↑↑↑ ALT, ↑↑↑ LD in serum and ALT > AST

• In obstruction, then only mild increases in serum AST, ALT, LD

Liver Enzyme Summary

• Most intense increase in ALP is seen in extra hepatic biliary obstruction.

• GGT is most sensitive for liver damage due to alcohol abuse.

• LD (specifically LD-5) is present in liver

Bilirubin Lab Measurement

Evelyn- Malloy Principle

• Bilirubin + Diazo Rgt → Azobilirubin

• Diazo Reagent = Sulfanilic acid + HCl + Sodium nitrite (NaNO3)

  • Conjugated (direct) bilirubin gives an immediate reaction

  • Unconjugated bilirubin must have albumin bond broken with methanol

Jandrasik-Grof Principle

• Bilirubin + Na acetate (buffer) + diazo

• Add ascorbic acid to stop reaction + Alkaline tartrate (changes pH)

• Read in spectrophotometer

  • Add caffeine-Na benzoate (accelerator) to measure indirect [unconjugated]

• DMSO [dimethyl sulfoxide] solubilizes the indirect (unconjugated) form of bilirubin

Porphyrin

• Normal ALA ↑ urorphyrinogen → Porphyria Cutanea Tarda ( PCT) → Most common

• ↑ ALA ↑ porphobilinogen in urine → Acute Intermittent Porphyria (AIP)

• ↑ ALA ↑ uroporphyrinogen and copro→ Cogenital Erythropoetic Porohyria( Gunther’sdisease)

• Protophyrin accumulates in the bone marrows,rbc, no excess porphyrins in urine → Erythropoetic Protoporphyri(EPP)

Laboratory - Watson- Schwartz Urobilinogen Erhlich’s test

• P-dimethylaminobenzaldehyde (Erhlich’s Reagent) + Na acetate

• Forms a red color with urobilinogen

• assessing liver function and detecting hemolytic diseases.

Plasma Protein Indicators of Nutritional Status

• Albumin

• Transferrin

• Transthyretin

  • (thyroxinebinding pre-albumin,

    pre-albumin)

• Retinol-binding Protein

• Insulin-like Growth factor, somatomedin

Albumin Half Life & Function:

• Half-Life: 18-20 days

• Maintains osmotic pressure and is carrier of

  hydrophobic molecules in plasma

Transferrin Half Life & Function:

• Half-Life: 2-3 days

• Function: Carrier protein for thyroid hormones in plasma,

  carrier for retinol-binding protein

Transthyretin Half Life & Function:

• Half-Life: 2-3 days

• Function: Carrier protein for thyroid hormones in plasma,

  carrier for retinol-binding protein

Retinol-binding Protein Half Life & Function:

• Half-Life: 12 hrs

• Function: Transports vitamin A in plasma; binds noncovalently to pre-albumin

Insulin-like Growth factor, somatomedin

• Half-Life: 2-6 hrs

• Function: Have similar metabolic effects to insulin; sensitive to nutritional variation while free from the effects of inflammation

Fat-Soluble Vitamins

• Vitamin A: Retinol, Retinal, Retinoic acid

• Vitamin D: Ergocalciferol, Cholecalciferol

• Vitamin E: Tocopherols, Tocotrienols

• Vitamin K: Phylloquinone, Menaquinones

Vitamin A:

• Function: Required for normal vision and immune function

• Disease:

  • Deficiency: degeneration of eyes and skin

  • Toxicity: abdominal pain, headaches, skin roughness

• Laboratory Assessment: Fluorospectrophotometry,

  immunoassay, HPLC

Vitamin D

• Function:

  • Calcium and phosphorous metabolism

  • Maintains bone structure

• Disease:

  • Deficiency: rickets, osteomalacia

  • Toxicity: hypercalcemia

• Laboratory Assessment: Immunoassay, HPLC

Vitamin E

• Function:

  • Antioxidant of unsaturated fatty acyl groups of membrane phospholipids

  • Protects membranes

• Disease:

  • Deficiency: hemolytic anemia due to fragility of RBC membranes

  • Toxicity: antagonistic to vitamin K, may enhance the effect of coumarin therapy, resulting in hemorrhage

• Laboratory Assessment: Erythrocyte hemolysis

  functional test, GC, HPLC

Vitamin K

• Function:

  • Required for synthesis of clotting factors II, VII, IX and X

  • Coumarins act by interfering with the activation of

    Vitamin K

• Disease: hemorrhagic disease, increased clotting time

• Laboratory Assessment: Immunoassay, HPLC,

  Prothrombin time functional test

Water-Soluble Vitamins

• Vitamin B1: Thiamine

• Vitamin B2: Riboflavin

• Vitamin B6: Pyridoxine

• Niacin: Nicotinic acid

• Folate

• Vitamin B12: cyanocobalamin

• Biotin

• Pantothenic acid

• Vitamin C: Ascorbic acid

Vitamin B1

• Function: Co-enzyme of metabolic reactions

• Disease: Deficiency - Beriberi

• Laboratory Assessment: Fluorospectrophotometry, HPLC, transketolase functional test

Vitamin B2

• Function: Component of coenzymes, FMN and

  FAD, for redox reactions

• Disease: Deficiency → General metabolic defect

• Laboratory Assessment: Fluorospectrophotometry, HPLC, glutathione reductase functional test

Vitamin B6

• Function: co-enzyme of metabolic reactions

• Disease: Deficiency → General metabolic defect

• Laboratory Assessment: HPLC, tyrosine decarboxylase functional test

Niacin

• Function: Component of coenzymes, NAD and

  NADP, for redox rxns

• Disease: Deficiency → Pellagra

• Laboratory Assessment: Fluorospectrophotometry

Folate

• Function: Carrier of one carbon groups for metabolic reactions

• Disease: Deficiency → Megaloblastic

  anemia

• Laboratory Assessment: Competitive Binding

  Protein, HPLC

Vitamin B12

• Function:

  • Complexes with intrinsic factor to

    pass through the

    intestinal mucosa

  • Involved in the

    synthesis of

    methionine and

    conversion of

    methylmalonate to

    succinate

• Disease: Deficiency → Megaloblastic anemia, Pernicious anemia

• Laboratory Assessment: Competitive Binding

  Protein, Immunoassay

Biotin

• Function: Prosthetic group for carboxylation reactions

• Disease: Deficiency → vomiting, anorexia, dermatitis

• Laboratory Assessment: Microbiological

  functional assay

Pantothenic acid

• Function: Component of carrier molecules such co-enzyme A and acyl protein carrier

• Disease: general metabolic defect

• Laboratory Assessment: Microbiological

  functional assay, immunoassay, GC, HPLC

Vitamin C

• Function:

  • Cofactor of protocollagen Aid the synthesis of cartilage, dentin, and bone

  • Reducing agent for hydroxylation

    reactions

• Disease: Scurvy, inability to form connective tissue

• Laboratory Assessment: Fluorospectrophotometry,

  photometry

Trace Element

Indicator of Nutritional Status

• Chromium

• Copper

• Selenium

• Zinc

Chromium

• Function: Promotes insulin action

• Disease: Glucose intolerance

• Laboratory Assessment: AAS

Copper

• Function: Component of redox reactions and cytochrome reactions

• Disease:

  • Menkes’ syndrome

  • Wilson’s disease

  • Indicated in inflammatory

    rxns

• Laboratory assessment:

  • AAS

  • Functional assay of superoxide dismutase and cytochrome c oxidase

Selenium

• Function: protects against oxidative stress constituent of enzymes

• Disease: Associated with increased instance of

  cancer and cardiopathy

• Laboratory Assessment: AAS

Zinc

• Function: Constituent and co-factor of enzymes

• Disease: General metabolic defect, including growth retardation and poor wound healing

• Laboratory Assessment: AAS

Enzyme Tumor Markers

• Prostate-specific antigen

• Lactate dehydrogenase

• Alkaline phosphatase

• Neuron-specific enolase

Tumor Marker: Tumor Types

• Prostate-specific antigen: Prostate Cancer

• Lactate dehydrogenase: Hematologic malignancies

• Alkaline phosphatase: Metastatic carcinoma of bone, hepatocellular carcinoma, osteosarcoma, lymphoma, leukemia

• Neuron-specific enolase: Neuroendocrine tumors

Tumor Marker: Method

• Prostate-specific antigen: IA

• Lactate dehydrogenase: EA

• Alkaline phosphatase: EA

• Neuron-specific enolase: RIA, IHC

Specimen for Enzyme Tumor Markers

Serum

Prostate-specific antigen: Clinical Utility

Prostate cancer screening, therapy monitoring, and recurrence

Lactate Dehydrogenase: Clinical Utility

Prognostic indicator; elevated nonspecifically in numerous cancers

Alkaline phosphatase: Clinical Utility

Determination of liver and bone involvement; nonspecific elevation in many bone-related and liver cancers

Neuron-specific enolase: Clinical Utility

Prognostic indicator and monitoring disease progression for neuroendocrine tumors

Carbohydrate and Cancer Antigen Tumor Markers

CA 19-9, CA 15-3, CA 27-29, CA-125 are biomarkers used to monitor certain cancers, including pancreatic, breast, and ovarian cancers.

CA 19-9, CA 15-3, CA 27-29, CA-125 Methods and Specimen

Immunoassay and Serum

CA 19-9, CA 15-3, CA 27-29, CA-125 Tumor Types

• CA 19-9: Gastrointestinal cancer and adenocarcinoma

• CA 15-3: Metastatic breast caner

• CA 27-29: Metastatic breast carcinoma

• CA-125: Ovarian Cancer

CA 19-9 Clinical Utility

Monitoring pancreatic cancer