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• Plasma proteins and enzymes include albumin (60%), globulin (35%), and fibrinogen (4%), with others at less than 1%.

Amino Acids

• Consist of an amino group, hydrogen, carboxylic acid group, alpha carbon, and an R-group.
• They are the building blocks of proteins.
• L-forms are naturally found in humans.
• There are 20 amino acids classified by polarity and charge properties.
• Hydrophobic amino acids have nonpolar R-groups and are usually found in the interior of proteins or at the surface where they interact with lipids.
• Hydrophilic amino R-groups form hydrogen bonds and are found on the surface where they interact with water.

Proteins

• Polypeptide chains are formed when amino acids join via peptide bonds (primary structure).
• Primary protein structure is the amino acid sequence.
• Secondary protein structure includes coiled alpha-helices and beta-pleated sheets formed by hydrogen bonding between peptide bonds.
• Tertiary protein structure is the folding of a protein into a compact, three-dimensional shape stabilized by interactions between side R-groups.
• Quaternary protein structure is a combination of two or more protein subunits forming a larger, biologically active protein.

Protein Digestion and Metabolism

• In the stomach, hydrochloric acid and pepsin digest proteins into peptides.
• In the intestines, bicarbonate, trypsinogen, chymotrypsinogen, proteolastase, procarboxypeptidase A and B are released by the pancreas and small intestine.
• Aminopeptidases in the blood break down peptides into di- and tripeptides and amino acids.
• These products pass through the epithelial cells lining the intestine.
• Absorbed amino acids are delivered to cells and reassembled into proteins in the liver.
• R-groups can be used for making carbohydrates or for lipid storage.
• In a fasting state, amino acids are metabolized for energy or used for gluconeogenesis.
• Amino group catabolism results in the excretion of urea.
• Nitrogen from amino acids is excreted as urea in urine.
• Nitrogen balance is indicated when the amount of nitrogen ingested equals the amount excreted.

Plasma Proteins

• Proteins are present in all body fluids and are frequently used for diagnostics.
• Intracellular enzymes detected in plasma provide a sensitive, non-specific indicator of tissue damage.
• Function in transportation, including transferrin, haptoglobin, lipoproteins, and hemoglobin; also function as enzymes.
• Function as intracellular enzymes include AST, ALT, LDH, and ALP.
• Function in immunity, including immunoglobulins and in the maintenance of oncotic pressure, mainly by albumin.
• Act as clotting factors such as coagulation factors and complement proteins.
• Function in inflammation as acute phase reaction proteins such as CRP and antitrypsin.
• Total protein levels include: CSF (<450 mg/dL), serum (60 to 90 g/L), and plasma (63 to 93 g/L).
• Total protein concentration depends on the rate of synthesis, rate of removal, and distribution volume.
• A rapid change in the total plasma protein concentration always results from changes in distribution volume.

Oncotic Pressure

• Osmotic pressure exerted by proteins in plasma or interstitial fluid maintains water levels inside blood vessels.
• Oncotic pressure is fairly stable, and changes will result in the body trying to restore balance.
• Increased water inside the blood vessel leads to hydrostatic pressure exerted on the interior membrane.

Hydrostatic Pressure

• Hydrostatic pressure is pressure of blood on the membrane that causes fluid to leave blood capillaries into interstitial space.
• Oncotic pressure counters hydrostatic pressure by pulling liquid into capillaries.

Relationship Between Osmotic Pressure and Hydrostatic Pressure

• When water moves into the blood vessel: osmotic pressure decreases, oncotic pressure decreases, hydrostatic pressure increases, and volume distribution increases.
• When water moves out of the blood vessels: osmotic pressure increases, oncotic pressure increases, hydrostatic pressure decreases, and volume distribution decreases.

Capillary Permeability

• Impacts on the movement of proteins and solutes with changes on oncotic and hydrostatic pressure by controlling movement of molecules across membranes.
• Larger molecules are kept on the right side of the membrane.
• Normal homeostasis in arterioles includes net flow out, decreased hydrostatic pressure, and increased oncotic pressure.
• There is no net flow in Capillary Bed.
• Venules include net flow in, with lower hydrostatic pressure and higher oncotic pressure.

Decreased Oncotic Pressure

• Hydrostatic pressure is higher than oncotic pressure at the capillary bed, resulting in increased fluid in the interstitial space known as water retention or edema.
• A typical sign of edema occurs when excess fluid builds up, causing swelling where pressure produces a pit or indentation.

Total Protein

• Consists of albumin and globulins (alpha-1, alpha-2, beta, and gamma globulins).
• Hyperproteinemia is an overall increase in protein values indicating dehydration or conditions synthesizing immunoglobulins.
• This could also be artefactual, caused by extended application of tourniquet during venipuncture, increased synthesis of globulin, dehydration, and decreased volume of distribution.

Hypoproteinemia

• Results from decreased albumin synthesis or increased volume distribution, as in overhydration or increased capillary permeability.
• Results from increased loss, excretion, degradation, hemorrhage, sepsis, fever, or malignant disease. Also indicated by the presence of a negative nitrogen balance.
• May result from decreased albumin synthesis (malnutrition, malabsorption, liver disease), overhydration (increased volume of distribution), or increased capillary permeability.
• Hypoalbuminemia is due to decrease in oncotic pressure with decreased low back into the vessels.

Hypoalbuminemia

• Albumin is the most abundant plasma protein contributing to 80% of oncotic pressure.
• Decreased albumin causes decreased oncotic pressure, resulting in decreased flow back into the vessels and accumulation of interstitial fluid known as edema.
• Albumin is found in serum, CSF, urine, interstitial fluid, and amniotic fluid.
• Plays a major role in maintaining of oncotic pressure, blood volume, pressure and balance of interstitial fluid level in tissues
• Functions as a transport protein for unconjugated bilirubin and some thyroid hormones..
• Increased level of albumin is caused by dehydration.
• Decreased level of albumin is caused by/results from malnutrition malabsorption, liver disease, renal loss.

Globulins

• Consist of alpha-1, alpha-2, beta, and gamma globulins.
• In serum, gamma globulins are account for the highest fraction.
• Gamma globulins are the immunoglobulins in our immune system.
• Hypoglobulinemia results in low Ig.
• In babies, low native Ig levels make them susceptible to infections.
• X-linked agammaglobulinemia results in almost complete absence of immunoglobulin.
• Dysgammaglobulinemia is a defect in ½ immunoglobulin, most commonly IgA deficiency, caused by secondary CLL, multiple myeloma, Hodgkin's disease, cytotoxic drugs, or nephrotic syndrome.
• Hypergammaglobulinemia results from physiological responses to an infection or to a specific antigen or autoimmune diseases, indicated by polyclonal or monoclonal antibodies (only 1 antibody produced in large quantity, A paraprotein is an immunoglobulin produced or by a single of cells of the B lymphocytes).

Major Plasma Enzymes

• Their measurements aid in the diagnosis and management of diseases.
• Enzymes with physiological function i.e. coagulation factors.
• Enzymes that have no physiological function i.e. when these enzymes are present as a result of cellular turnover.
• Lactate dehydrogenase and aspartate transaminase are elevated in heart attack or liver disease.
• CK (creatine kinase) elevations indicate a heart attack.
• ALT (alanine transaminase) is elevated in hepatitis.
• ALP (alkaline phosphatase) is elevated in liver or bone disease.
• LPS and amylase are elevated in pancreatic disease.
• ACP (acid phosphatase) is elevated in prostate carcinoma.

Multiple Myeloma

• Cancer of plasma cells that leads to excessive growth and malfunction in the bone marrow.
• Cancerous plasma cells produce paraprotein (monoclonal immunoglobulin), increasing plasma paraprotein concentration.
• Bone destruction occurs due to osteoclast-activating cytokines (e.g., IL-1, TNF-β) released by tumors.
• Hypercalcemia results from excessive bone breakdown.
• Paraprotein and calcium can obstruct the nephrons, leading to renal failure.
• Bence Jones Proteins (light chains with low molecular weight) are secreted by the tumor and rapidly cleared into urine. Occur in 75% of cases.
• 20% of patients do not have a paraprotein band on serum electrophoresis but have Bence Jones Proteins.
• β2-microglobulin (>6 mg/L) suggests renal impairment and increased tumor activity.
• Paraproteinemia can replace plasma water with protein, leading to low sodium levels (hyponatremia).
• Plasma cell overgrowth in the bone marrow interferes with the production of RBCs, WBCs, and platelets, causing anemia.
• Bone pain, destruction, and fractures mainly affect lumbar vertebrae, ribs, and pelvis, that may lead to numbness or paralysis.

Proteinuria

• Normal limit for protein in urine: ≤150 mg/day.
• Abnormal proteinuria occurs when proteins exceed this threshold due to kidney dysfunction.
• In systemic conditions, multiple myeloma increases Bence-Jones proteins (low molecular weight), intravascular hemolysis increases hemoglobin in urine.
• Low-molecular-weight proteins (<70 kDa) filtered by the glomerulus are not reabsorbed due to tubular damage.
• Glomerular proteinuria is caused by increased glomerular permeability, allowing high-molecular-weight proteins to be filtered into urine, creating heavy proteinuria (up to 5 g/24h), and filtered proteins are metabolized in tubules, preventing their appearance in urine by glomerulonephritis, systemic lupus erythematosus (SLE), and diabetic nephropathy.
• Tubular proteinuria results from damage to renal tubules that prevent reabsorption of low-molecular-weight proteins (e.g., β2-microglobulin), cause mile proteinuria, and increases in urinary β2-microglobulin indicate renal tubular damage.
• Orthostatic (Postural) Proteinuria occurs as benign condition that is caused by increased hydrostatic pressure in renal veins when standing. That only appears after prolonged standing and and does not affect the kidney.
• Microalbuminuria is the presence of a small concentration of albumin in urine that cannot be found with normal dipstick tests. It is a sign for glomerular damage, ESRD, and retinopathy in diabetics, with a normal amount of albumin being less than 30 mg/24h and over 30-300 mg/24h indicates microalbuminuria.

Cerebrospinal fluid (CSF) Protein

• Proteins in the CSF originate from plasma by crossing the blood-brain barrier (BBB).
• Normal CSF protein range is 0.15 – 0.45 g/L (up to 0.9 g/L in neonates and elderly).
• Causes of Increased CSF Protein:
  • Meningeal Inflammation (Meningitis)
  • Increased capillary permeability, allowing proteins to cross the BBB into CSF.
  • Tumors can disrupt normal CSF dynamics, leading to protein accumulation.
  • Oligoclonal bands (OCBs) in CSF indicate immune system involvement in MS.

Multiple Sclerosis

• A central nervous system disorder marked by decreased nerve function with initial inflammation of the protective myelin nerve covering and eventual scarring with alternating episodes of crisis and remission.
• Increased IgG/Albumin Ratio (Normal: ~10%; In MS: Increased to 50%) indicates the disease.
• Oligoclonal Bands (OCBs) where electrophoresis of CSF shows oligoclonal bands in the y-globulin region produced by B-cell clones and indicates intrathecal (CNS) IgG production.
• For Specimen collection use Gel separator tube for Total Protein & Albumin Measurement.
• Serum is preferred over plasma because plasma contains fibrinogen, which forms a β2 region band, potentially mimicking a paraprotein. Proteins migrate based on net negative charge, forming distinct bands.

Serum Protein Electrophoresis (SPEP)

• A process that is not widely used except for detecting paraproteins or to identify monoclonal antibody production, as is the can in B-cell tumors.