Clinical Chem Quiz 1

Essential amino acids

1. Arginine

2. Histidine

3. Isoleucine

4. Leucine

5. Lysine

6. Methionine

7. Phenylalanine

8. Threonine

9. Tryptophan

10. Valine

Gold standard for IEM screening

HPLC Tandem Mass Spec

Plasma

Cell free portion of blood that includes coagulants

Serum

Portion of blood that has no coagulants (especially fibrinogen)

>2 peptide link protein measurement

1. Proteins added to reagent with copper sulfide and alkalines

2. Copper breaks down links and creates color change

3. Color change is measured at 540nm

   1. Not sensitive to protein <0.1mg/dL

Major serum proteins

• Prealbumin (transthyretin)

• Albumin

• Alpha 1 antitrypsin

• Alpha 2 macroglobulin

• Beta 2 transferrin

• Gamma immunoglobulins (G, A, M, CRP)

Nephrotic syndrome SPEP profile

• Kidneys passing too much protein, increase in alpha 2 molecules to make up and keep up oncotoic balance
  
  

Polycloncal gammapothy

• Increase in gamma globulins, many types

Monocolonal gammopathy

• increase in one type of monoclonal antibody

Hypogammaglobinemia

• Lack of gamma globulins

Acute phase inflammation response INCREASES IN…

• C-reative protein

• Procalcitonin

• Haptoglobin

• Fibrinogen

• ALpha-1-trypsin

• Ceruloplasmin

• Ferritin

• Serum amyloid A

Acute phase inflammation response DECREASE IN…

• Albumin

• Transferrin

• Prealbumin

• Retinol binding protein

• Alpha-fetoprotein

Albumin

• Contributes to oncotic pressure and fluid balance

• 3.5 to 5.2 g/dl

• Marker for nutrition status and liver capacity

• Heavy and negative charged

• Measured through Bromocresol green dye binding

Microalbumin (urine albumin)

• Albumin in urine (should be low)

• >300mg/day is considered positive (BAD)

• Measured via turbidity or nephelometry

• 24 hour urine collection is preferred

Prealbumin (transthyretin)

• Good marker for nutrition status

• Transports thyroxine and retinol

• Normal range 18-39 mg/dl

• Measured via turbidity of nephelometry

Anti-tryspin 1

• Inhibits proteases from breaking down self proteins (protects liver and lungs from neutrophil breakdown)

• Can lead to emphysema (damage to alveoli) or liver disease

  • 
    

Haptoglobin

• Found in serum

  • Marker for hemolysis (acute phase reactant)

Ceruloplasmin

• Copper transport protein

• Can leads to Wilson disease (defect in ceruloplasmin binding copper, leading to excess)

  • Manifests as Fleischer rings in eyes

    • High liver copper amounts, low serum levels and high urine levels

• Can leads to Menkes‘ disease (defect in copper transport)

  • Manifests as neurodegenerative disorders

    • Low liver copper

Transferrin

• Binds to ferric iron (fe3+)

• Measures via turbidity

• Presents as high transferrin in Iron Deficiency Anemia

Ferritin

• Stores iron

• Measures via sandwich immunoassay

• Presents as low ferritin in Iron Deficiency Anemia

• Presents as high ferritin in Iron Overload

B2 transferrin

• Marker for CSF

• Detection via immunofixation

B2 microglobulin

• Marker for kidneys

• Measures renal tubular disorders (low serum levels, high urine levels), kidney heavy metal damage, multiple myeloma)

C-reatice protein

• <10 mg/L indicates low probability of bacterial infection

Phenylketonuria

• Missing Phenylalanine hydroxylase (PAH)

• Body cannot break down phenylalanine, leading to toxic build up

  • Treated via diet change

Alkaptonuria

• Build up of homogentisic acid (by product of alanine to tyrosine), leading to black urine

  • Treated via diet change

Maple syrup urine disease

• Body cannot breakd down branched amino acids leading to maple-syrup-smelling urine

• Treat with low protein diet, consider liver transplant

Cofactor

Inorganic non protein that is required for enzyme activity

Coenzyme

Organic protein or molecule that is required for enzyme activity

Enzyme function

Biomolecule that catalyzes (speeds up) a reaction

Rate of catalyzed reaction

Vmax

The max rate of a reaction

kM (Michelis constant)

Substrate concentration at ½ the Vmax

Zero and first order

First order - exponential portion of reaction, not dependent on substrate concentration

Zero order - plateauing portion of reaction where enzymes are occupied

Enzyme present assay

• We put excess substrate to get to V max and measure

or

• We put enzyme in excess so that V is proportional to concentration of substrate

or

• We add a ton of substrate and let it run to completion

Competitive inhibitors

Binds to active site (decreases the amount of enzyme available)

kM increases, Vmax stays the same

Non competitive inhibitor

Binds to somewhere on the active site to (slows it down)

kM stays the same, Vmax decreases

Uncompetitive inhibitor

Binds to the substrate enzyme complex

Enzyme activity constant

1. End point method (stop the reaction at a fix time, measure absorbance of initial vs final)

   1. Kinetic method (where we have multiple measurements at specific time intervals)

Enzyme activity measurement

IU

or

Umol/min

or

Umol/min/L

Creatine Kinase/Phosphatase (CK) source, function and diagnosis

Source: Highest around the skeletal muscle, heart and brain

Function: Creatine-P to Creatine

Diagnosis: Increase = source tissue disorder

Creatine Kinase/Phosphatase (CK) measurement and error source

Measurement: Creatine P to creatine + ATP, leads to NADH with 2 enzymes

Error: Hemolysis

CK isoenzymes

CK-MM (Skeletal muscle)

CK-BB (Brain) (>500U/L is severe, >205U/L) is death

CK-MB (Heart)

Lactate dehydrogenase (LDH) Source and diagnosis and function

Source: Heart, liver, skeletal muscles

Elevations: Tissue cell and inflammation

Function: Lactate to pyruvate

GREAT marker for reinforcing diagnosis

LDH measurement and error

Measurement: LActate to pyruvate creates NADH which is measurable

Error: Hemolysis

Aspartate aminotransferase (AST) source, elevation and measurement

Source: heart>liver>skeletal muscle>RBCs>serum

Elevation: liver damage (moderate = cirrhosis, large = toxic hepatitis)

Measurement: AST, then use MD to create NAD which is measurable

AST Error

Source: hemolysis (very much so), only stable for 3 to 4 days when refrigerated

Alanine aminotransferase (ALT) Source and diagnosis

Source: Many tissues but high concentration in the liver

Elevation: high ALT and AST? liver disease, normal ALT, high AST? non-liver source, AST/ALT ratio >3? alcoholic hepatitis or cirrhosis

ALT error and measurement

Error: Stable for 3-4 days, unaffected by hemolysis

Measurement: Reaction coupled with LDH to generate NAD

Alkaline phosphate ALP source, diagnosis and function

Source: Liver, bone, spleen, placeneta

Diagnosis: Hepatobiliary disorder, bone disorder

Function: Catalyzes phosphate monoester cleavage

ALP error and measurement

Measurment: removal of phosphate from P-nitrophenylphosphate, produces P-nitrophenolate which is yellow at 405NM

Error: hemolysis

y-Glutamyltransferase GGT source, diagnosis

Source: Liver, bile ducts, kidney, brain, pancreas

Diagnosis: hepatobiliary disoder, alcoholism, pancreatitis, diabetes mellitus

Measurement: Production of P-nitroanaline which can be measured at 405NM

If AST, ALT greater than ALP, GGT then…

• Primary liver disease, acute hepatitis or jaundice

If ALP, GGT greater than ASP or ALT then…

• Obstructive liver disease or jaundice, post hepatic jaundice or hepatobiliary disorder

Glucose -6-phosphate dehydrogenase G6PD source, function and diagnosis

Source: adrenal cortex, spleen, thymus, lymph nodes, mammary glands

Function: G6P to 6Phosphogluconate with release of NADPH

Diagnosis: Toxic drug reactions and heinz bodies

G6PD measurement

Measurement: Hemolysate

Macroenzymes

• High molecular mass forms of enzymes in serums (bound to Ig)

• High activity detected as they are too big to be passed through kidney

Purpose of hexose-monophosphate shunt

Generation of NADPH and precursors to nucleotides

Glucose reference ranges

UW 62-125mg/dL

Hypoglycemia

• Low blood glucose (less than 62mg/dL)

• Manifests as hunger, sweating, nausea

Hyperglycemia

• Increase in plasma glucose caused by imbalance of hormones (>125mg/dL)

• Symptoms exhibited at >200mg/dL

  • Polyuria (lots of pee)

Fasting plasma glucose

• When you have no foods or drinks 8-12 hours before a test

  • Normal fasting: 70-99mg/dL

  • Impaired fasting 100-125mg/dL

  • Provisional diabetes >126mg/dL

Type 1 diabetes mellitus effect and risks

Hyperglycemia caused by damaged pancreas makes little to no insulin (moves sugar to blood stream into cells)

• Autoantibodies attack spleen Beta-cells that make insulin

  • Leads to excess glucagon

  • Risk factors: family history, infection, virus

Type 2 diabetes mellitus effect and risks

• Greater tendency to get into coma

• Family history, obesity, unhealthy diet

  • Insulin resistance: often obese, high blood pressure, dark velvety skin patches

    • Fasting glucose >100mg/dL

Gestational diabetes mellitus

• Gestational

  • Placental estrogen and cortisol block the mother's insulin leading to insulin resistance and hyperglycemia

  • Treat by changing diet

• Done at 24 to 48 weeks

Gestational diabetes mellitus tests

Glucose challenge

Oral glucose

Glucose challenge test

Administer 50g of glucose, measure serum glucose after 1 hour

• Normal <140mg/dL, abnormal >140mg/dL

Oral glucose test

Pregnant patient fasts for 8-14 hours

Take baseline plasma glucose then administer 75g in 250-300mL

• Normal < 140mg/dL, impaired glucose tolerance 140-199mg/dL, diabetes >200mg/dL

Criteria of diabetic diagnosis

• HBA1C > 6.5%

• Random glucose level >200mg/dL

• Fasting plasma glucose >125mg/dL

• Oral glucose tolerance test >200mg/dL after 2 hours after taking 75g glucose load

Metabolic syndrome

• Hypertension

• Increased serum glucose

• Abnormal lipids

• Increased body fat

Risk factor, often those with it have diabetes

Specimen tubes

• Lime green (Plasma separation tube)

• Orange (Rapid serum tube)

• Gold (Serum separation tube)

• Grey sodium fluoride/potassium oxalate (glycolysis prevention)

• Red top (no preservatives)

Hemoglobin A1C test

reflects 2-3 months of glycemic control

Normal: 4-5.6%, prediabetic: 5.7-6.4%

Requires lavender top

Hemoglobin A1C test methods

• HPLC and boronate affinity chromatography

  • Seperates different hemoglobin species

• A1c immunoassay

  • Monoclonal antibody to glucose attaches to glycated hemoglobin beta chain

• Enzyme assay

  • Cleaves N terinal valine, oxidation and colormetric detection

Glucose oxidase reaction

Hexokinase reaction

Lactate measurement

Type 1 von gierke disorder

• Glucose 6 phosphatase deficiency

  • Can't make glycogen

type 3 Cori disease

Deficiency of gylcogen debrancher

Type 4 Amylopectinosis

Deficiency of branching enzyme, abnormal glycogen build up

Galactosemia

• Inability to metabolism galactose

  • Can't metabolism galactose to lactose leading to build up of galactose in blood

• Leads to brain damage, cataracts, jaundice, kidney damage