BIOCHEMICAL INVESTIGATION OF BLOOD PLASMA PROTEINS

Total protein content of normal plasma is 6 to 8 g/ 100 ml (65-85 g/l). As a first step of study, the plasma proteins may be separated into Albumin (35-50g/l), Globulins (25-35 g/l) and fibrinogen (2-4 g/l).

The albumin : globulin ratio is usually between 1.2:1 to 1.5:1.

Almost all plasma proteins, except immunoglobulins are synthesized in liver.

In clinical laboratory, total proteins in serum or plasma of patients are estimated by Biuret method.

In clinical laboratory, electrophoresis is employed regularly for separation of serum proteins.

 The term electrophoresis refers to the movement of charged particles through an electrolyte when subjected to an electric field.

The positively charged particles (cations) move to cathode and negatively charged ones (anions) to anode. Since proteins exist as charged particles, this method is widely used for the separation of proteins in biological fluids. The technique was first used by Tiselius in 1937; named it as moving boundary or frontal electrophoresis (Nobel Prize in 1948).

Factors Affecting Electrophoresis

The rate of migration (separation of particles) during electrophoresis will depend on the following factors:

1. Net charge on the particles

2. Mass and shape of the particles.

3. The pH of the medium.

4. Strength of electrical field.

5. Properties of the supporting medium.

6. Temperature.

Types of Electrophoresis

There are mainly two types of electrophoresis—horizontal and vertical.

Different types of support media are used in horizontal electrophoresis, e.g. filter paper, cellulose acetate, agar gel, agarose gel, starch gel, etc. The vertical electrophoresis mainly uses polyacrylamide gel. The nature of the supporting medium will also influence the mobility.

Electrophoresis Apparatus

The electrophoresis system basically consists of the electrophoresis tank to hold the buffer and fitted with the electrodes, as well as a power pack to supply electricity at constant current and voltage. When the electrophoresis is carried out, the buffer is  chosen in such a way so as to ensure effective separatum of the mixture of proteins. The pH and ionic strength and nature of the buffer may be varied according to the proteins to be separated, e.g. serum proteins are separated at a  pH of 8.6 using barbitone buffer. At this pH all serum proteins will have a net negative charge and will migrate towards the anode.                                

Support Medium for Electrophoresis  

Filter Paper                                    

Cellulose Acetate Membrane

Agar or Agarose

Starch Gel

Polyacrylamide gel electrophoresis (PAGE)

Visualisation of Protein Bands

After the electrophoretic run is completed, the proteins are fixed to the solid support using a fixative such as acetone or methanol. Then it is stained by using dyes

(Amido Schwartz, naphthalene black, Ponceau S or Coomassie Blue) and then destained by using dilute acetic acid. The electrophoretogram can be scanned using a densitometer and each band quantitated. In the densitometer, light is passed through the agar gel plate; the absorption of light will be proportional to the quantity of protein present on a band. Another method is that the stain may be eluted from the support and each fraction quantitated colourimetrically.

 

Normal Values and Interpretations

In agar gel electrophoresis, normal serum will be separated

into five bands. Their relative concentrations are given

below:

Albumin                                 :  55-65%

Alpha-1 -globulin                  :     2-4%

Alpha-2-globulin                   :    6-12%

Beta-globulin                         :    8-12%

Gamma-globulin                    :   12-22%

 

ALBUMIN

The name is derived from the white precipitate formed when egg is boiled (Latin, albus = white). Albumin constitutes the major part of plasma proteins. It has one polypeptide chain with 585 amino acids and 17 disulfide bonds. It has a molecular weight of 69,000. It is synthesised by hepatocytes; therefore, albumin level in blood is decreased in liver cirrhosis. Estimation of albumin is a liver function test. Half-life of albumin is about 20 days. Liver produces about 12 g of albumin per day, representing about 25% of total hepatic protein synthesis. Albumin can come out of vascular compartment. So albumin is present in CSF and interstitial fluid. Over 80 allelic forms of albumin, all with normal function, but with altered electrophoretic mobilities are described. Albumin is coded by a gene on the long arm of chromo­some number 4.

Functions of Albumin

1. It contributes to the colloid osmotic pressure of plasma.

The total osmolality of serum is 278-305 m osmol/ kg (about 5000 mm of Hg). But this is produced mainly by salts, which can pass easily from intravascular to extravascu lar space. Therefore, the osmotic pressure exerted by electrolytes inside and outside the vascular compartments will cancel each other. But proteins cannot easily escape out of blood vessels, and therefore, proteins exert the 'effective osmotic pressure'. It is about 25 mm Hg, and 80% of it is contributed by albumin. The maintenance of blood volume is dependent on this effective osmotic pressure. According to Starling's hypothesis, at the capillary end the blood pressure (BP) or hydrostatic pressure expels water out, and effective osmotic pressure EOP) takes water into the vascular compartment. At arterial end of the capillary, BP is 35 mm Hg and EOP is 25 mm; thus water is expelled by a pressure of 10 mm Hg. At the venous end of the capillary, EOP is 25 mm and BP is 15 mm, and therefore water is imbibed with a pressure of 10 mm. Thus, the number of water molecules escaping out at arterial side will be exactly equal to those returned at the venous side and therefore blood volume remains the same. If protein concentration in serum is reduced, the EOP is correspondingly decreased. Then return of water into blood vessels is diminished, leading to accumulation of water in tissues. This is called edema. Edema is seen in conditions where albumin level in blood is less than 2g / dl.

2.                           Another major function of albumin is to transport
various hydrophobic substances.

3.                            Being a watery medium,
blood cannot solubilise lipid components. Bilirubin and
non-esterified fatty acids are specifically transported by
albumin. Drugs (sulpha, aspirin, salicylates, dicoumarol,
phenytoin), steroid hormones, thyroxine, calcium, copper
and heavy metals are non-specifically carried by albumin.
Only the unbound fraction of drugs is biologically active.
The histidine residue at position 3 of albumin binds
copper.

4.                                                    All proteins have buffering capacity. Because of its high concentration in blood, albumin has maximum buffering capacity. Albumin has a total of 16 histidine residues which contribute this buffering action.

5.                  All tissue cells can take up albumin by pinocytosis. It is then broken down to amino acid level. So albumin may be considered as the transport form of essential amino acids from liver to extrahepatic cells.

Clinical Applications

        1. Albumin-fatty acid complex cannot cross blood-brain barrier and hence fatty acids cannot be taken up by brain. The bilirubin from albumin maybe competitively replaced by aspirin and such other drugs. In newborns, bilirubin is already high, and if such drugs are given, there is a probability that free bilirubin is deposited in brain leading to kernicterus and mental retardation.

2.          When two drugs having high affinity to albumin are administered together, there may be competition for the available sites, with consequent displacement of one drug. Such an effect may lead to clinically significant drug interactions, e.g. phenytoin -dicoumarol interaction.

3.          Protein-bound calcium is lowered in hypo­albuminemia. Thus, even though total calcium level in blood is lowered, ionised calcium level may be normal, and so tetany may not occur. Calcium is lowered by 0.8 mg/dl for a fall of 1 g/dl of Albumin.

4.          Human albumin is therapeutically useful to treat burns, hemorrhage and shock..

Normal Value and Interpretation: Normal level of Albumin is 3.5-5 g/dl. Lowered level of albumin (hypoalbuminemia) has important clinical significance.

Hypoalbuminemia

       1.   In cirrhosis of liver and in chronic liver failure, albumin

synthesis is decreased and so blood level is lowered.

2.         In malnutrition and in malabsorption syndrome the availability of amino acids is reduced and so albumin synthesis is affected.

3.         In nephrotic syndrome, kidney glomerular filtration is defective so that albumin is excreted in large quantities. Increased loss, to a certain extent, is compensated by increased synthesis; but blood level of albumin is decreased.

4.         Presence of albumin in urine (albuminuria) is always pathological. Large quantities (several grams per day) of albumin is lost in urine in nephrotic syndrome. Small quantities are lost in urine in acute nephritis, and other inflammatory conditions of urinary tract. Detection of albumin in urine is done by heat and acetic acid test. In microalbuminuria or minimal albuminuria or pauci-albuminuria, small quantity of albumin (50-300 mg/ d) is seen in urine (Paucity = small in quantity). However, microalbuminuria is also clinically important, as it is a predictor of future renal diseases.

5.         In burns, albumin is lost through the unprotected skin surface.

6.         In protein losing enteropathy large quantities of albumin is lost through intestines.

7.         Hypoalbuminemia will result in tissue edema. It may be seen in malnutrition, where albumin synthesis is depressed {generalized edema), in nephrotic syndrome, where albumin is lost through urine {facial edema) or in cirrhosis of liver (mainly ascites). In chronic congestive cardiac failure, venous congestion will cause increased hydrostatic pressure and decreased return of water into capillaries and so pitting edema of feet may result.

8.         Albumin is a negative acute phase protein; level of albumin falls mildly in presence of inflammatory cytokines such as interleukin-6.

9.         Analbuminemia {absence of albumin) is a very rare genetically determined condition.

Albumin-Globulin Ratio: In all the above mentioned conditions of hypoalbuminemia, there will be a compen­satory increase in globulins which are synthesised by the reticuloendothelial system. Albumin-globulin ratio (A/G ratio) is thus altered or even reversed. This again leads to edema.

Hypoproteinemia: Since albumin is the major protein present in the blood, any condition causing lowering of albumin will lead to reduced total proteins in blood (hypoproteinemia). So it is observed in cirrhosis, nephrotic syndrome, malnutrition and malabsorption syndromes.

Hyper-gamma -globulinemias

        1. When  albumin  level  is decreased, body tries to compensate it by increasing the production of globulins from reticuloendothelial system. Thus, all causes for

hypoalbuminemia will result in albumin : globulin ratio reversal, and corresponding increase in percentage in globulins.

2.          In chronic infections, the gamma globulins are increased, but the increase is smooth and widebased.

3.          Drastic increase in globulins are seen in para­proteinemias, when a sharp spike is noted in electro­phoresis. This is termed as M-band. This is due to monoclonal origin of immunoglobulins in multiple myeloma.

Hyper-beta-globulinemia: It is associated with hyper­lipoproteinemia, atherosclerosis and other hyperlipidemic conditions.

Hyper-alpha-globulinemia: In nephrotic syndrome, small molecular weight proteins (including albumin) leak out through urine. But proteins with larger molecular weight remains in blood; so there is an increase in alpha globulin fraction, which contains alpha-2-macroglobulin.

TRANSPORT PROTEINS

Blood is a watery medium; so lipids and lipid soluble substances will not easily mix in the blood. Hence such molecules are carried by specific carrier proteins. Albumin is an important transport protein, which carries bilirubin, free fatty acids, calcium and drugs.

1.    Pre-albumin is so named because of its faster mobility in electrophoresis than albumin. It is more appropriately named asTransthyretin or Thyroxin binding pre-albumin (TBPA), because it carries thyroid hormones, thyroxin (T4) and tri-iodothyronine T3). It can bind loosely with all substances which are carried by albumin. Its molecular weight is lesser than that of albumin. It is rich in tryptophan. Its half-life in plasma is only one day.

2.    Retinol binding protein (RBP) carries vitamin A. It is a low molecular weight protein, and so is liable to be lost in urine. To prevent this loss, RBP is attached with pre-albumin; the complex is big and will not pass through kidney glomeruli. It is a negative acute phase protein. Zinc is required for RBP synthesis, and so RBP level and vitamin A level may be lowered in zinc deficiency.

3.  Thyroxine binding globulin (TBG) is the specific carrier molecule for thyroxine and tri-iodo thyronine. TBG level is increased in pregnancy; but decreased in nephrotic syndrome.

4.  Transcortin, otherwise known as Cortisol binding globulin (CBG) is the transport protein for Cortisol and corticosterone..

5.  Haptoglobin (for haemoglobin), Hemopexin (for heme) and Transferrin (for iron) are importantto prevent loss of iron from body.

6.  Cholesterol in blood is carried by lipoproteins, HDL and LDL varieties.

 

ACUTE PHASE PROTEINS

The level of certain proteins in blood may increase 50 to 1000-folds in various inflammatory and neoplastic conditions. Such proteins are acute phase proteins. Interleukins (ID, especially IL-1 and IL-6, released by macrophages and lymphocytes, are the primary agents which cause induction and release of these acute phase proteins. Important acute phase proteins are C-reactive protein, ceruloplasmin, haptoglobin,a1 -acid glycoprotein, a-1-anti-trypsin and fibrinogen.

C-reactive Protein (CRP):

It is thus named because it reacts with C-polysaccharide of capsule of pneumococci. CRP consists of five polypeptide subunits to form a disc-shaped cyclic polymer. It has a molecular weight of 115-140 kD. It is synthesised in liver. It can stimulate complement activity and macrophage phagocytosis. When the inflammation has subsided, CRP quickly falls. CRP level has a positive correlation in predicting the risk of cardiovascular disease.

Ceruloplasmin

Ceruloplasmin is blue in colour (Latin, caeruleus=blue). It is an alpha-2 globulin with molecular weight of 160,000. It is synthesised in liver. It contains 6 to 8 copper atoms per molecule. Ceruloplasmin is also called Ferroxidase, an enzyme which helps in the incorporation of iron into transferrin. Ninety per cent of copper content of plasma is bound with ceruloplasmin, and 10% with albumin. Copper is bound with albumin loosely, and so easily exchanged with tissues. Hence transport protein for copper is Albumin. Ceruloplasmin is an enzyme. It is an important antioxidant in plasma.

Clinical Application:

 Normal blood level of ceruloplasmin is 25-50 mg/dl. It is estimated either by its oxidative property on phenylene diamine, or by radial immunodiffusion. This level is reduced in Wilson's hepato­lenticular degeneration. Ceruloplasmin level less than 20 mg/dl is pathognomonic of Wilson's disease. It is an inherited autosomal recessive condition. Incidence of the disease is one in 50,000. The defect is associated with chromosome No.13. The basic defect is a mutation in a gene encoding a copper binding ATPase in cells, which is required for excretion of copper from cells. So, copper is not excreted through bile, and hence copper toxicity is seen. Increased copper content in hepatocyte inhibits the incorporation of copper to apo-ceruloplasmin. So ceruloplasmin level in blood is decreased. Accumulation in liver leads to hepatocellular degeneration andcirrhosis. Deposits in brain basal ganglia leads to lenticular degeneration and neurological symptoms. Another common finding is copper deposits as green or golden pigmented ring around cornea; this is called Kayser-Fleischer ring. Copper deposits in kidney may cause renal failure, and in bone marrow leads to hemolytic anemia. Treatment consists of a diet containing low copper and injection of D-penicillamine which excretes copper through urine. Since zinc decreases copper absorption, zinc is useful in therapy.

Lowered levels of ceruloplasmin is also seen in malnutrition, nephrosis, and cirrhosis. Ceruloplasmin is an acute phase protein. So its level in blood may be increased in all inflammatory conditions, collagen disorders and in malignancies.

Alpha-1 Anti-trypsin (AAT)

AAT is otherwise called a-anti-proteinase or protease inhibitor (Pi). It inhibits all serine proteases (proteolytic enzymes having a serine in their active centre), such as plasmin, thrombin, trypsin, chymotrypsin, elastase, and cathepsin. Serine protease inhibitors are abbreviated as Serpins. Binding of this inhibitor to protease is very tight; once bound it is not released. Normally, about 95% of the anti-protease activity in plasma is due to AAT. It is synthesised in liver. It has a molecular weight of 50,000 and has 3 polypeptide chains. It forms the bulk of moleculesjn serum having a-1 mobility. It is estimated by radial immuno-diffusion method. Normal serum level is 75-200 mg/dl. Electrophoretically, multiple allelic forms can be separated, the most common variety is PiMM determined by the genotype MM. More than 75 variants are known, out of which about 30 genetic variants show decreased or very low serum concentrations. Gene is located on the small arm of chromosome number 14.

 

AAT deficiency causes the following conditions:

1. Emphysema: The deficiency is inherited as a co-dominant trait. The incidence of AAT deficiency is 1 in 1000, and is one of the most common inborn errors. About 5% of total population carry the abnormal gene in heterozygous state. The total activity of a1-AT is reduced in these individuals. Bacterial infections in lung attract macrophages which release elastase. In the a1-AT deficiency, unopposed action of elastase will cause damage to lung tissue, leading to emphysema. About 5% of emphysema cases are due to a1-AT deficiency. The alpha-1 band on electrophoresis is reduced or absent. The methionine residue at 358 position of a1-AT is important in the enzyme binding. This methionine may be oxidised to methionine sulfoxide by smoking. So emphysema is very common in smokers with normal a1-AT level and smoking will worsen the situation in a1-AT deficient persons.

2.     Cirrhosis: AAT deficiency is also seen in persons with PiZZ genes. This genetic make up is associated with cirrhosis of liver. The ZZ protein has a substitution of glutamic acid by lysine at position 342. The protein is unsialylated and is not released from hepatocytes, causing death of cells with consequent fibrosis and cirrhosis.

3.     In Nephrotic syndrome, AAT molecules are lost in urine, and so AAT deficiency is produced.

Alpha-2-Macroglobulin (AMG): AMG is a tetrameric protein with molecular weight of 725,000. It is the major component of a-2 proteins. Gene is located in the long arm of chromosome number 12. It is synthesised by hepa­tocytes and macrophages. AMG inactivates all proteases,  and thus it is an important in vivo anti-coagulant. Proteases cleave the "bait" region of AMG, releasing a small unit, to provoke conformational changes in AMG, which then "traps" the enzyme. So proteolytic enzymes cannot function. AMG-protease complexes are internalised by a receptor mediated endocytosis by macrophages, and then degraded. AMG is the carrier of many growth factors such as platelet derived growth factor (PDGF). Normal serum level is 130^300 mg/dl. AMG contributes about 1% of all total plasma proteins. Its concentration is markedly increased (up to 2-3 g/d I) in Nephrotic syndrome, because other proteins are lost through urine in this condition.

Alpha-1-Acid Glycoprotein: It is otherwise known as Orosomucoid. It has a molecular weight of 44,000 and has a high content of about 45% of carbohydrates. Its isoelectric pH is 0.7-3.5. It is synthesised by hepatocytes. It binds lipophilic substances and various drugs. It binds with progesterone tightly. Normal serum level is 55-140 mg/dl, and its half-life is five days. It is increased in pregnancy. It is also an acute phase protein. It is a reliable indicator of clinical activity of ulcerative colitis.

NEGATIVE  ACUTE  PHASE  PROTEINS

During an inflammatory response, some proteins are seen to be decreased in blood; these are called negative acute phase proteins. Examples are albumin, transthyretin (pre­albumin), retinol binding protein and transferrin.

Transferrin: It is a specific iron binding protein. It is a negative acute phase protein; so the blood level is decreased in acute diseases. It has a half-life of 7-10 days and is used as a better index of protein turnover than albumin.

Evaluation of proteinuria.

Proteinuria means the excretion of protein in the urine. A healthy person does not excrete proteins in the urine or the excretion of proteins is less than 150 mg per day. The proteins most commonly found in the urine are those derived from the plasma of blood and consist of a mixture of albumin and globulin. Predominantly albuminuria (excretion of albumin in urine) is detectable on routine urine analysis during a medical examination. Albuminuria could be organic (due to involvement of kidneys or other organs) or functional (due to physiological or biological stress on kidneys). The functional albuminuria is usually intermittent and not accompanied by any symptoms or evidence of kidney disease. Renal function tests and urinary deposits are found to be normal during the functional albuminuria. It may be connected with posture; being absent when the person is lying down and present when standing. The functional albuminuria usually clears up in early adult life and seems to be associated with the growth and development of kidneys. Any severe stress may also lead to transient albuminuria. Exposure to severe cold and excessive exercise or physical activity may cause functional or transient proteinuria. However, there is nothing to worry about as the functional albuminuria is self limiting with respect to the cause. Mild to moderate functional albuminuria may also be detected during last two months of pregnancy due to pressure on kidneys.

Organic albuminuria is of three types: 1) Renal Albuminuria - When the cause is the kidney disease. 2) Pre-renal Albuminuria - When the kidneys are affected secondarily to some other disease. Post-renal Albuminuria - When the protein is added to the urine after it has left the renal tubules.

1.     Renal Albuminuria: It is found in all forms of kidney disease. The cause of renal disorder or kidney disease may be inflammatory (infectious), degenerative (immunological) or destructive (toxic or malignant). The plasma globulin and red blood cells (RBCs) may also be excreted along with albumin during some renal disorders. The urine would be smoky in color if macroscopic hematuria (blood in urine) is also associated with proteinuria. The cases of acute glomerulonephritis may excrete 0.5 to 2.0 percent (0.5 g to 2.0 g/dl) protein in the urine, whereas the cases affected by chronic glomerulonephritis generally excrete less than 0.5 percent (0.5 g/dl) protein in the urine. The amount of protein excreted daily would vary depending on the volume of urine voided daily. The ratio of albumin to globulin excreted in the urine may vary from 10:1 to 5:1. A routine and quantitative urine analysis is required to evaluate the extent of excretion of proteins in the urine.

2.     Pre-renal Albuminuria: It is found in a variety of conditions exerting stress on the kidneys. The pre-renal albuminuria usually disappears when the primary disease is cured. Impairment of renal circulation due to dehydration, diarrhea or vomiting, blood loss due to accidental injuries or anemia are the most common conditions, which could lead to pre-renal albuminuria.

3.     Post-renal Albuminuria: The proteinuria or albuminuria is termed as post-renal albuminuria if protein is possibly added to the urine as it passes along the urinary tract after leaving the urinary tubules of the kidneys. The major causes of the post-renal albuminuria are the lesions of the renal pelvis or urinary bladder. Lesions of the prostate (in male patients) and urethra also lead to post-renal albuminuria. Admixture of discharges from the vagina (in female patients) and semen (in male patients) may also give positive tests for protein.

People with diabetes, hypertension, or certain family backgrounds are at risk for proteinuria. In the United States, diabetes is the leading cause of ESRD.1 In both type 1 and type 2 diabetes, albumin in the urine is one of the first signs of deteriorating kidney function. As kidney function declines, the amount of albumin in the urine increases.

Another risk factor for developing proteinuria is hypertension, or high blood pressure. Proteinuria in a person with high blood pressure is an indicator of declining kidney function. If the hypertension is not controlled, the person can progress to full kidney failure.

African Americans are more likely than Caucasians to have high blood pressure and to develop kidney problems from it, even when their blood pressure is only mildly elevated. In fact, African Americans are six times more likely than Caucasians to develop hypertension-related kidney failure.2

Other groups at risk for proteinuria are American Indians, Hispanics/Latinos, Pacific Islander Americans, older adults, and overweight people. These at-risk groups and people who have a family history of kidney disease should have their urine tested regularly.

Proteinuria has no signs or symptoms in the early stages. Large amounts of protein in the urine may cause it to look foamy in the toilet. Also, because protein has left the body, the blood can no longer soak up enough fluid, so swelling in the hands, feet, abdomen, or face may occur. This swelling is called edema. These are signs of large protein loss and indicate that kidney disease has progressed. Laboratory testing is the only way to find out whether protein is in a person’s urine before extensive kidney damage occurs.

Several health organizations recommend regular urine checks for people at risk for CKD. A 1996 study sponsored by the National Institutes of Health determined that proteinuria is the best predictor of progressive kidney failure in people with type 2 diabetes. The American Diabetes Association recommends regular urine testing for proteinuria for people with type 1 or type 2 diabetes. The National Kidney Foundation recommends that routine checkups include testing for excess protein in the urine, especially for people in high-risk groups.

The evaluation of proteinuria is shown schematically in and is typically initiated after colorimetric detection of proteinuria by dipstick examination. The dipstick measurement detects mostly albumin and gives false-positive results when pH > 7.0 and the urine is very concentrated or contaminated with blood. A very dilute urine may obscure significant proteinuria on dipstick examination, and proteinuria that is not predominantly albumin will be missed. This is particularly important for the detection of Bence Jones proteins in the urine of patients with multiple myeloma. Tests to measure total urine concentration accurately rely on precipitation with sulfosalicylic or trichloracetic acids. Currently, ultrasensitive dipsticks are available to measure microalbuminuria (30 to 300 mg/d), an early marker of glomerular disease that has been shown to predict glomerular injury in early diabetic nephropathy.

Until recently, an accurate protein measurement required a 24-hour urine collection. In a 24-hour collection, the patient urinates into a container, which is kept refrigerated between trips to the bathroom. The patient is instructed to begin collecting urine after the first trip to the bathroom in the morning. Every drop of urine for the rest of the day is to be collected in the container. The next morning, the patient adds the first urination after waking and the collection is complete.


Containers for a 24-hour urine collection.

In recent years, researchers have found that a single urine sample can provide the needed information. In the newer technique, the amount of albumin in the urine sample is compared with the amount of creatinine, a waste product of normal muscle breakdown. The measurement is called a urine albumin-to-creatinine ratio (UACR). A urine sample containing more than 30 milligrams of albumin for each gram of creatinine (30 mg/g) is a warning that there may be a problem. If the laboratory test exceeds 30 mg/g, another UACR test should be done 1 to 2 weeks later. If the second test also shows high levels of protein, the person has persistent proteinuria, a sign of declining kidney function, and should have additional tests to evaluate kidney function.


Cup for a single urine sample.

The magnitude of proteinuria and the protein composition in the urine depend upon the mechanism of renal injury leading to protein losses. Large amounts of plasma proteins normally course through the glomerular capillaries but do not enter the urinary space. Both charge and size selectivity prevent virtually all of albumin, globulin, and other large-molecular-weight proteins from crossing the glomerular wall. However, if this barrier is disrupted, there can be leakage of plasma proteins into the urine. Smaller proteins (<20 kDa) are freely filtered but are readily reabsorbed by the proximal tubule. Normal individuals excrete less than 150 mg/d of total protein and only about 30 mg/d of albumin. The remainder of the protein in the urine is secreted by the tubules (Tamm-Horsfall, IgA, and urokinase) or represents small amounts of filtered b2-microglobulin, apoproteins, enzymes, and peptide hormones. Another mechanism of proteinuria occurs when there is excessive production of an abnormal protein that exceeds the capacity of the tubule for reabsorption. This most commonly occurs with plasma cell dyscrasias such as multiple myeloma and lymphomas that are associated with monoclonal production of immunoglobulin light chains.

The normal glomerular endothelial cell forms a barrier penetrated by pores of about 100 nm that holds back cells and other particles but offers little impediment to passage of most proteins. The glomerular basement membrane traps most large proteins (>100 kDa), while the foot processes of epithelial cells (podocytes) cover the urinary side of the glomerular basement membrane and produce a series of narrow channels (slit diaphragms) to allow molecular passage of small solutes and water. The channels are coated with anionic glycoproteins that are rich in glutamate, aspartate, and sialic acid, which are negatively charged at physiologic pH. This negatively charged barrier impedes the passage of anionic molecules such as albumin. Some glomerular diseases, such as minimal change disease, cause fusion of glomerular epithelial cell foot processes, resulting in predominantly "selective" (Fig. 2) loss of albumin. Other glomerular diseases can present with disruption of the basement membrane and slit diaphragms (e.g., by immune complex deposition), resulting in large amounts of protein losses that include albumin and other plasma proteins. The fusion of foot processes causes increased pressure across the capillary basement membrane, resulting in areas with larger pore sizes. The combination of increased pressure and larger pores results in significant proteinuria ("nonselective").

When the total daily excretion of protein exceeds 3.5 g, there is often associated hypoalbuminemia, hyperlipidemia, and edema (nephrotic syndrome). However, total daily urinary protein excretion greater than 3.5 g can occur without the other features of the nephrotic syndrome in a variety of other renal diseases . Plasma cell dyscrasias (multiple myeloma) can be associated with large amounts of excreted light chains in the urine, which may not be detected by dipstick (which detects mostly albumin). The light chains produced from these disorders are filtered by the glomerulus and overwhelm the reabsorptive capacity of the proximal tubule. A sulfosalicylic acid precipitate that is out of proportion to the dipstick estimate is suggestive of light chains (Bence Jones protein), and light chains typically redissolve upon warming of the precipitate. Renal failure from these disorders occurs through a variety of mechanisms including tubule obstruction (cast nephropathy) and light chain deposition.