CENTRAL TEXAS COLLEGE 2401 - CLINICAL CHEMISTRY Amino Acids and Proteins I. Amino Acids A. Basic structure 1.

Small molecules in which carbon attached to: a. one amino group (-NH2) b. one carboxyl group c. R group 1) Side chain which determines amino acid 2) Example a) glycine R group = -H b) alanine R group = -CH3 2. Subunits used for making proteins B. Metabolism 1. 20 amino acids for human protein production a. 9 essential amino acids supplied from diet b. Body synthesizes other amino acid. c. Breakdown of body proteins amino acids Form amino acid pool: used for synthesis of body proteins

2. II.

Aminoacidopathies A. Phenylketonuria (PKU) 1. Definition -- condition caused by deficiency in or absence of enzyme phenylalanine hydroxylase 2. Clinical significance a. Incidence 1 in 10,000 live births b. Congenital, inherited defect (homozygous) c. Due to increase phenylalanine concentration (>20 mg/dL) in blood, normally minor pathways of phenylalanine metabolism are activated, leading to increased production of phenylketones (phenylpyruvic acid) d. Brain damage and mental retardation 1) Occurs 2 - 3 weeks after birth 2) Due to an accumulation of PKU metabolites via alternate pathways 3. Phenylalanine metabolism a. Normal 1) Phenylalanine converted to tyrosine (irreversible reaction) 2) Tyrosine is precursor compound in: a) Thyroid hormones b) Fumaric acid and acetoacetic acid (TCA intermediates) c) Adrenal medulla hormones and melanin

b.

Abnormal 1) Phenylketonuria (PKU) 2) Thyroid dishormogenesis 3) Hypopigmentation 4) Alkaptonuria

4.

Prevention of PKU a. Early detection b. Low phenylalanine diet 1) Milk - high in phenylalanine 2) Artificial sweetener: aspartame - high in phenylalanine Laboratory determinations a. Blood, serum or plasma 1) Fluorometric -- phenylalanine is reacted with ninhydrin in the presence of the dipeptide L-leucyl-L-alanine to form a fluorescent product which is % to the phenylalanine conc. 2) HPLC of phenylalanine 3) Guthrie test -- utilization of phenylalanine by Bacillus subtilis b. Urine for phenylpyruvic acid 1) Urine - to monitor progress of disease 2) Not used for screening sample of infants because conc.s must reach 12-15 mg/L, which does not occur until 10-14 days after birth 3) Ferric chloride test a) Principle -- ferric ions will react with phenylpyruvic acid to yield a blue-green color. b) Procedure (1) Positive -- blue-green color (2) Negative -- no change (yellow) c) Sources of error (1) Acetoacetic acid -- red color (2) Salicylates -- purple color

5.

B.

Alkaptonuria 1. Definition a. Rare hereditary metabolic disease b. Deficiency of HGA oxidase which converts tyrosine to fumarate and acetoacetic acid c. Results in accumulation of homogentistic acid (HGA) 2. Clinical Significance a. Causes the urine to turn dark on standing b. HGA in connective tissue pigmentation of tissues and arthritis-like degeneration

C.

Cystinuria 2

1.

2.

Definition a. Hereditary condition b. Excessive urinary excretion of cysteine and three other dibasic amino acids c. Due to defect in renal resorptive mechanism Clinical Significance a. Manifested by formation of urinary cysteine calculi (kidney stones)

D.

Homocystinuria 1. Definition a. Inborn error in metabolism of sulfur amino acid.s b. Defect in pathway of methionine cysteine c. Lack of enzyme - cystathionine synthase d. Requires cofactor B6 2. Clinical Significance a. Brain damage and mental retardation b. Skeletal deformities c. Excrete large amounts of homocysteine and methionine in urine Maple Sugar Urine Disease (MSUD) 1. Definition a. Inborn error blocks metabolism of 3 essential branched chains amino acids: 1) leucine 2) isoleucine 3) valine b. Lack of enzyme - branched chain ketoacid decarboxylase c. leucine, isoleucine, and valine found in blood, urine, and CSF 2. Clinical Significance a. Burnt sugar odor of urine, breath, and skin b. Hypoglycemia and acidosis c. Mental retardation and convulsions d. Classical disease usually results in death within first year of life

E.

III.

Proteins A. General characteristics 1. Structure a. Composed of elements C, O, H, N, and S (no N atoms in carbs or lipids) b. Macromolecules composed of covalently linked polymers of amino acids c. Peptide bond - carboxyl group of one amino acid. combines with amino group of another amino acid. resulting formation of covalent bond and formation of water molecule 2. Charge and Isoelectric point (pI) 3

3.

Amphoteric (can bear + and - charges) because of free terminal carboxyl and amino groups b. Net charge of molecule depends of pH c. Isoelectric point - pH at which amino acid./protein has no net charge (# of positively charged groups = # of negatively charged groups) Immunogenicity a. Effective as antigens, substances that induce an immune response b. Elicit antibody production when injected into animals of another species

a.

B.

Anabolism/Catabolism 1. Nitrogen balance - balance between protein anabolism and catabolism 2. Anabolism (synthesis) - synthesized in the liver and secreted by hepatocyte into the circulation 3. Catabolism - mostly in the liver a. protein --- amino acids b. amino acid.s ---deaminated-- ammonia + ketoacids c. ammonia - urea and excreted in urine d. ketoacids (Kreb's cycle) -- glucose or fat 4. 2 major groups a. Simple proteins - yield only amino acid. b. Conjugated proteins - protein plus a non-protein group Measurement 1. Electrophoresis - best method for assessment of serum proteins 2. Separation by salt a. As salt conc. increases, proteins become dehydrated and less soluble (salting out) b. Different proteins precipitate at different salt concentrations 3. Separations can also be accomplished by ultracentrifugation, gel filtration, chromatography, and immunochemistry Functions 1. Transport a. Nutrients b. Hormones c. Waste d. Drugs/toxins 2. Buffer systems 3. Colloid osmotic pressure determinant 4. Immunoglobulins 5. Coagulation 6. Connective tissue 7. Biocatalysts Clinical significance 1. Reference total protein conc.: 6.5 - 8.3 g/dL 4

C.

D.

E.

2.

3.

Decreased values a. Starvation b. Over hydration c. Renal disease d. Malabsorption syndrome Increased values a. Infections b. Dehydration

F.

Measurement of total protein 1. Qualitative assays (amino acids) a. Ninhydrin reaction -- specific for amino acids or free amine groups b. Microbiological assays -- presence of specific amino acids are indicated by growth of microorganisms which cannot synthesize particular amino acid in question c. UV (280 nm) absorbance -- due mostly to phenylalanine and tyrosine residues 2. Quantitative tests a. Biuret -- measures peptide bonds 1) Principle -- biuret reagent, which contains Cu++ ions, reacts with protein in an alkaline medium to form a reddishpurple complex. Intensity of color is % to protein conc. and is read at 540 nm b. Other techniques 1) Kjeldahl technique -- based on measurement of protein nitrogen (assumed to be 16% of total mass) 2) Dye Binding most proteins in serum bind dye use Coomassie brilliant blue 250 and see shift in abs from 465 to 595 3) Refractive index -- optical measurement by refractometer

IV.

Plasma Proteins A. Prealbumin (Transthyretin) 1. Moves ahead of albumin during electrophoresis 2. Binds thyroxine (T4) and triiodothyronine (T3); transport for thyroid hormones and Vitamin A 3. levels of prealbumin with inflammation, malignancy and cirrhosis of liver; sensitive marker for poor protein nutritional status 4. prealbumin with Hodgkin's disease, alcoholism, chronic renal failure Albumin 1. Most prevalent plasma protein produced in liver

B.

2.

Function a. Transports/binds 1) Long chain fatty acids 5

b. c. d. 3.

2) Bilirubin 3) Cortisol 4) Aldosterone 5) Thyroxine Binds toxic, heavy metals Maintains plasma colloid osmotic pressure Reserve store of amino acids

Clinical significance a. Reference values: 3.5-5.5 g/dL b. Increased values 1) Dehydration 2) Stasis c. Decreased values 1) Malnutrition 2) Chronic kidney disease 3) Extensive burns 4) Infections 5) Impaired synthesis - liver disease Albumin assay a. Principle -- albumin reacts with bromcresol green (BCG) dye to form a green complex. Intensity of color is % to albumin conc. and is read at 630 nm b. Green color is result of a change in color of dye from a yellowish-green to a deeper green c. pH of solution is critical. BCG is a pH indicator and will change color as pH shifts d. Method has a high specificity for albumin e. Other dye binding procedures use: 1) 2-(4'-hydroxyazobenzene)-benzoic acid (HABA) 2) bromcresol purple (BCP) 3) methyl orange

4.

C.

Globulins 1. Types a. Produced in the liver 1) -1 Anti-trypsin a) Neutralizes enzymes that cause damage to structural proteins b) Low levels associated with neonatal respiratory distress syndrome, severe lung disease c) Elevated activity with inflammatory reactions, pregnancy and contraceptive use d) Acute phase reactant a protein that is markedly increased in plasma during acute phase of inflammatory process 2) Lipoprotein--transport complex fats 3) Haptoglobin--binds free hemoglobin 4) Transferrin--carries iron in the blood 6

5) 6) 7) 8) 9) 10) b.

-2 Macroglobulin--inhibit proteases -fetoprotein Ceruloplasmin Hemopexin Fibrinogen C-reactive protein

Immunoglobulins 1. IgG 2. IgA 3. IgM 4. IgD 5. IgE

D.

Albumin -- Globulin Ratio (A/G Ratio) 1. Not a specific indicator of any specific disease 2. Disease states alter the A/G ratio which is normally 1.3:1 to 1.8:1 (reported as 1.3 or 1.8 A/G ratio) 3. Calculation Albumin Total Globulins = A/G ratio or Albumin (Total Protein - Albumin) a. Albumin = 4.0 g/dL (from a BCG procedure) b. Globulin = 2.5 g/dL (calculated from difference of Total Protein and Albumin values) c. A/G = 4.0 2.5 = 1.6:1 = 1.6 A/G ratio Fibrinogen 1. Function-- precursor to fibrin, the actual clot forming fiber in the clotting mechanism 2. Clinical significance a. Reference plasma values -- 200-400 mg/dL b. Decreased values 1) Afibrinogenemia -- genetic problem, no synthesis 2) Dysfibrinogenemia -- a defective protein chain whose activity has been altered. Results in abnormal clot formation 3) Low fibrinogen levels -- plasmin deficiencies 4) Liver disease 5) DIC a) Premature separation of the placenta -- almost immediate drop in fibrinogen levels b) Amniotic fluid embolism c) Retention of dead fetus d) massive injury e) surgery c. Increased levels 1) Tissue damage 2) Inflammatory diseases 7

E.

3) 3.

a) Rheumatic fever b) Pneumonia c) Septicemia d) Tuberculosis Pregnancy

Chemical assays a.. Biuret method -- addition of calcium and thrombin overcomes effect of anticoagulant. Fibrin clot is removed, redissolved with heat and NaOH, and biuret procedure is performed on that specimen b. Electrophoresis -- fibrinogen appears as a peak between and fractions

F.

Immunoglobulins 1. Clinical significance a. Hypergammaglobulinemia 1) Monoclonal gammopathy -- increase in one molecular species of immunoglobulin arising from a neoplasm of clones of same lymphocyte a) Multiple myeloma -- rapid, uncontrolled growth of Ab producing plasma cells. These malignant tumors can produce any of classes of Ig. In . 60% of patients, light chain production is increased and found in urine as BenceJones proteins b) Waldenstrom's macroglobulinemia -- monoclonal tumor produces IgM. Due to high molecular weight of protein, it creates a high blood viscosity c) Benign tumors (1) Most often IgG (2) Rarely become malignant 2) Polyclonal gammopathy -- Ig's are produced by clones of many plasma cells with an increase of many of immunological classes. IgG normally rises most significantly 3) Systemic Lupus Erythematosus (SLE) -- an autoimmune, inflammatory disease occurring primarily in women of childbearing age. Blood vessels are attacked by the body's own immune system. Characterized by elevated IgD concentrations b. Hypogammaglobulinemias 1) Primary -- deficiency in immunoglobulin synthesis. Usually attributed to a genetic abnormality a) Transient hypogammaglobulinemia -- IgG levels diminish over 4-6 month period until there is no IgG or IgA. Respiratory infections are common b) Severe Combined Immune Deficiency (SCID or X-link infantile infections) 1 X chromosome abnormality 2 Unable to produce any Igs 3 Most common of severe inherited immune disorders 8

4 5 6 2)

Defects in B and T lymph function Frequent infection begin at 3-6 months old Usually fatal by end of 2nd year

Secondary - normal B-lymphs with IgG levels. IgA and IgM levels may/may not be decreased Acquired Immune Deficiency Syndrome - infection of T-lymphs which prevents immune response through blocking of immunological production (Retrovirus Human Immune Virus (HIV))

3)

c.

Assay 1) Electrophoresis/immunoelectrophoresis (qualitative) a) 12-22% of total proteins b) Majority in fraction 2) Immunodiffusion (quantitative) 3) Double diffusion (quantitative)

VI.

Proteins in Urine A. Normal protein reflects protein of 1. 2. 3. B. Plasma Renal tubular origin Genito-urinary tract (e.g., mucins)

Reference ranges 1. 2. 50 - 150 mg/24 hours Up to 25 mg/dL in random specimens

C.

Proteinuria 1. 2. 3. Definition -- presence of excess of serum protein in urine Transitory proteinuria -- healthy individuals Persistent a. Kidney disease 1) Pyelonephritis -- bacterial infection 2) Glomerulonephritis -- streptococcal infection 3) Nephrosis 4) Renal hypertension 5) Tumors b. Urinary tract infection 1) Bacterial, fungal or parasitic 2) Increased proteins, especially mucins c. Exogenous causes 1) Disease elsewhere in body 9

2) 3) D. Bence-Jones Protein 1. 2. 3. 4. 5. 6.

Elevated plasma proteins Cleared by the kidney

Abnormal protein Diagnostic of the disease multiple myeloma Occurrence: approx. 50% of all multiple myeloma cases Molecular weight: 45,000 amu 25% of total -globulins Bence-Jones test a. Principle: 1) Upon addition of acetate buffer (pH 4.9) and heat, Bence-Jones protein will precipitate at 40 - 60 OC and redissolve above 60 OC 2) With cooling, it reprecipitates at 40 - 60 OC and then redissolves at room temperature Interpretation: 1) Precipitation at 40 - 60 OC Positive Bence-Jones protein 2) Redissolution above - 60 OC

b.

E.

Mucins 1. Mucoproteins (glycoproteins) 2. From genito-urinary tract 3. Small amounts are normally present (2 - 5 mg/dL) a. Normal concentrations are NEG during routine testing b. Elevations 1) POS on qualitative testing 2) Indicate infection 3) False POS tests, may result from presence of reducing substances Proteoses 1. Protein fragments 2. Migrate electrophoretically with the -2 globulins 3. Not precipitated by heat 4. Faintly precipitated with TCA Qualitative Tests 1. Sulfosalicylic acid test (SSA) a. Principle: Urine protein is precipitated by 3% sulfosalicylic acid. A visual observation is made of amount of turbidity produced b. Interpretation: 1) No turbidity -- negative 2) Slightly perceptible turbidity -- trace 3) Greater degrees of turbidity are: a) 1+ = approximately 50 mg/dL b) 2+ = turbidity with no granulation approx. 200 mg/dL 10

F.

G.

c) d) 2. 3.

3+ = turbidity with granulation and flocculation, approx. 500 mg/dL 4+ = clumps of precipitated protein, approx. 1 g/dL

Trichloroacetic acid (similar to SSA test) Heat and acetic acid a. Principle: Albumin and globulin are coagulated by heat and acid pH. Acetic acid is used to provide optimum pH (4-5) and sodium acetate provides inorganic salt for protein flocculation b. Interpretation: same as the SSA test Chemical Test (dipstick) a. Principle: "protein of indicators. 1) At fixed pH, tetrabromphenol blue will have one color in presence of protein and another color in absence of protein 2) Citrate buffer provides a hydrogen ion concentration of approx. pH 3 3) Tetrabromphenol blue indicator changes from yellow to green to blue in presence of increasing amounts of protein Interpretation: 1) No color change -- negative 2) Color change: yellow to green to blue, depending on concentration -- trace, 1+, 2+, 3+ and 4+

4.

b.

H.

Quantitative Tests for 24 Hour Urines 1. Trichloroacetic acid test a. Principle: addition of trichloroacetic acid (TCA) to urine specimen precipitates protein in fine suspension which is quantitated by turbidimetry at 620 nm b. Modification of TCA test uses supernatant of TCA-treated urine for blanking and absorbance is read at 420 nm after 35 min Biuret method a. Principle: peptide bonds of protein react with Cu(II) ions in alkaline solutions to form colored product whose ABS is measured at 540 nm b. Biuret reagent contains sodium potassium tartrate to complex cupric ions and maintains their solubility in alkaline solution c. Generally not sensitive enough for low protein concentrations found in urine Electrophoresis - urine is concentrated for assay; electrophoretic patterns interpreted for presence and becomes semiquantitative

2.

3.

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VI.

Proteins in Cerebrospinal fluid (CSF) A. Cerebrospinal Fluid (CSF) 1. Definition a. Colorless fluid surrounding and filling non-tissue spaces of brain b. Maintains pressure constancy and provides mechanical "water-jacket" protective coating

B.

CSF Protein 1. 2. > 80% of CSF protein content originates from plasma by ultrafiltration through walls of capillaries in meninges and choroid plexus CSF proteins (low molecular weight plasma proteins) a. Prealbumin b. Albumin c. Transferrin Remainder originates from intrathecal synthesis Lowest concentration of total protein and smallest proportion of larger protein molecules are in ventricular fluid CSF protein concentration increases as it passes down to lumbar spine Example of difference in conc. at different levels: a. Ventricular fluid: 5-15 mg/dL total protein b. Cisternal fluid: 15-25 mg/dL total protein c. Lumbar fluid: 15-45 mg/dL total protein

3. 4. 5. 6.

C.

Clinical significance 1. Used chiefly to detect increased permeability of blood/brain barrier to plasma proteins or to detect increased intrathecal secretion of Igs 2. Bacterial or viral meningitis -- exudative inflammation of meninges. a. protein b. glucose 3. Intracerebral or subarachnoid hemorrhage a. protein b. Glucose normal c. Appearance: bloody, xanthochromic, icteric 4. Spinal cord depression due to tumor or abscess (Froin's syndrome) a. Greatly protein, may show clotting b. Glucose normal 5. Other conditions which increase permeability of blood/brain barrier a. Encephalitis -- inflammation of the brain b. Poliomyelitis -- acute viral disease with involvement of CNS Test procedures 1. Samples a. Specimen -- lumbar puncture (spinal tap) 1) Can only be collected by physician 2) Problems encountered 12

D.

b.

Small amounts usually available Bloody tap contaminated with plasma constituents, may be confused with cerebral hemorrhage c) CSF must be considered EXTREMELY contagious and handled with extreme precaution Factors to examine 1) Physical examination 2) Cytological examination 3) Chemical examination a) Protein - reference range 15-45 mg/dL b) Glucose - reference range 50-80 mg/dL (60-75% of plasma glucose levels)

a) b)

2.

Method for determination of total protein in CSF a. Turbidimetric method b. Ultraviolet spectrophotometry c. Immunoglobulins (and other specific proteins) in CSF 1) Pandy test: qualitative turbidimetric method (measures globulin levels) 2) Radial immunodiffusion (measures specific proteins, i.e., IgG) 3) Electrophoresis - Oligoclonal banding a) Seen in 90% of Multiple Sclerosis patients b) High resolution technique required c) Multiple distinct bands in globulin zone

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