Kidney functions tests-Non-Protein Nitrogen (NPN)-Part I + II

What are non-protein nitrogen (NPN) compounds?

Substances that contain nitrogen but are not part of the protein molecules. They are the by products of proteins and nucleic acid catabolism.

What are some examples of NPN compounds?

1. Urea

2. Free Amino Acid

3. Uric Acid

4. Creatinine

5. Creatine

6. Ammonia

What is the primary function of NPN compounds in the body?

They monitor kidney function as they are excreted through the urine and can provide insights into renal health

How do NPN compounds help in monitoring kidney function?

Urea and creatinine are particularly essential for evaluating kidney function. The kidneys excrete these compounds, and elevated levels in the blood can indicate impaired kidney function or renal failure

Why are NPN compounds also used to monitor hepatic function?

Urea is made in the liver and monitoring these levels helps asses liver function because reduced urea synthesis can suggest liver dysfunction or damage

How are NPN levels used to assess nutritional status?

They are used to asses the nitrogen balance in the body, which provides insight into a person’s nutritional status.

What do nitrogen levels indicate

Positive nitrogen balance indicates adequate protein intake and retention, while negative nitrogen balance suggests protein breakdown or insufficient intake.

How do NPN levels indicate Kwashiorkor?

This is a condition caused by protein deficiency, high NPN levels may be observed due to increased protein breakdown

How do NPN levels indicate Marasmus?

This is a condition that results from both caloric and protein deficiency, low NPN levels may suggest overall nutritional deficiency

How can NPN levels serve as an indicator of nutritional status?

For example, elevated NPN levels may suggest excessive protein breakdown, while low levels may indicate inadequate nutrition. NPN tests are often used to diagnose and monitor conditions such as kwashiorkor and marasmus.

What is the role of NPN in understanding overall nutritional deficiencies?

Elevated NPN may indicate protein breakdown, while low NPN levels may point to inadequate nutrition, whether due to lack of protein or overall caloric intake.

How can NPN testing assist in managing patients with malnutrition?

It helps healthcare providers understand the severity of the nutritional deficiency and track the effectiveness of interventions aimed at improving protein and calorie intake.

What are the primary non-protein nitrogenous metabolites used for kidney function tests?

• Creatinine

• Urea

• Uric acid

Why are creatinine, urea, and uric acid used as kidney function tests?

Because they are waste products filtered and excreted by the kidneys. Their levels in the blood can indicate how well the kidneys are functioning, particularly in terms of glomerular filtration.

How do creatinine, urea, and uric acid help in measuring the glomerular filtration rate (GFR)?

A decrease in the GFR suggests impaired kidney function, as the kidneys are less able to filter out these metabolites.

How are NPN levels measured in clinical practice?

Using plasma or serum samples

What role do the kidneys and liver play in relation to NPN?

The kidneys remove NPN, while the liver makes NPN.

What is the bottom line regarding the use of creatinine, urea, and uric acid?

They are used as indicators of kidney function and other conditions (e.g., liver disease).

What is urea/BUN?

A major non-protein nitrogen-containing compound and a metabolic product of protein catabolism

What are the sources of protein that contribute to urea/BUN?

• Exogenous protein (diet)

• Endogenous protein

Where is urea synthesized?

In the hepatic enzymes in the Krebs and Henselheit urea cycle (liver).

What is another major product of the urea cycle?

arginine.

What percentage of urea/BUN is filtered from the plasma by the glomerulus?

90%

What percentage of urea is reabsorbed by passive diffusion?

40%

Why is urea not a good measure of GFR?

Because 40% is reabsorbed by passive diffusion, which affects its accuracy in estimating glomerular filtration rate.

What percentage of urea is excreted through the kidneys?

60%

What percentage of urea is excreted through the GI tract and skin?

Less than 10%

What is the main role of urea in the body?

A waste that accounts for 75-80% of non-protein nitrogen (NPN) excreted by the body.

What factors determine urea absorption?

• Concentration of urea (protein diet)

• Urine flow rate (strength of bladder)

• Degree of hydration of the person

What are the reference intervals for urea nitrogen in adults?

• In plasma or serum: 6–20 mg/dL (2.1–7.1 mmol/L)

• In urine (24-hour): 12–20 g/day (0.43–0.71 mol urea/day) or 430 to 710 mmol/day

What is the role of solvent drag in urea reabsorption?

It refers to the process by which urea follows water during reabsorption at the proximal convoluted tubule (PCT).

What happens to many glomerular filtrates at the PCT?

Many glomerular filtrates, including urea, are reabsorbed at the proximal convoluted tubule (PCT).

What factors does blood urea concentration depend on?

• Protein content of the diet

• Protein catabolism

• Liver function

• Renal function

  • Glomerular filtration rate (GFR)

  • Renal perfusion (blood flow to the kidney)

  • Level of hydration

How does protein content of the diet affect blood urea concentration?

Higher protein intake increases protein catabolism, which in turn raises urea production.

How does liver function influence blood urea concentration?

The liver is responsible for synthesizing urea. Impaired liver function can lead to reduced urea production, affecting its levels in the blood.

How does renal function impact blood urea concentration?

The kidneys are responsible for filtering and excreting urea. Impaired kidney function, particularly a decrease in glomerular filtration rate (GFR) or reduced renal perfusion, can cause an increase in blood urea levels.

What is the effect of reduced renal perfusion on blood urea concentration?

Reduced renal perfusion, such as in congestive heart failure (CHF), leads to decreased blood flow to the kidneys. This can result in an increase in blood urea nitrogen (BUN) levels.

How does the level of hydration affect blood urea concentration?

If a patient is less hydrated, their serum BUN will increase, while urine BUN will decrease, due to decreased kidney function and reduced ability to excrete urea efficiently.

What are the factors that determine urea reabsorption?

• Solvent drag (or bulk transport) – Urea, being a small molecule, follows water in the proximal convoluted tubule (PCT).

• Hydration state and BUN level – The concentration of BUN influences urea reabsorption.

How does hydration state affect urea reabsorption and BUN levels?

In a well-hydrated state, more BUN is excreted in the urine, resulting in a lower serum BUN. Hydration helps flush out BUN, essentially detoxifying the body.

What happens to urea reabsorption in a dehydrated state?

In a dehydrated state, the body conserves water. About 70% of the BUN is reabsorbed, and only 30% of the BUN is excreted. This leads to an increase in serum BUN and a decrease in urine BUN.

What is uremia?

It is a condition associated with severe kidney disease where various types of wastes and non-protein nitrogen (NPN) compounds increase. It involves an elevated concentration of plasma urea, BUN, and other waste products in the blood.

What is azotemia?

It refers to elevated nitrogenous compounds, such as BUN and creatinine, in the blood. It is a condition that indicates an increased nitrogen content in the body.

Why is the BUN/creatinine ratio measured?

To assess the cause of azotemia. It helps differentiate between various types of azotemia (pre-renal, renal, and post-renal).

What is pre-renal azotemia?

Due to reduced renal blood flow or decreased renal perfusion, such as in conditions like congestive heart failure (CHF) or a high protein diet. Despite elevated nitrogenous waste levels, kidney function is still normal in pre-renal azotemia.

What is renal azotemia?

Due to decreased renal function, specifically renal failure. In this condition, the glomerular filtration rate (GFR) is decreased, and less BUN is excreted in the urine, leading to an accumulation of BUN in the serum.

What is post-renal azotemia?

Due to an obstruction in the urine flow, either in the kidneys, bladder, or urethra. This blockage prevents normal excretion of waste, leading to an increase in BUN and other nitrogenous compounds in the blood.

What causes prerenal azotemia?

A reduced renal blood flow, such as in conditions like congestive heart failure (CHF), where there is reduced renal perfusion.

How does reduced renal perfusion affect urea delivery and filtration?

It leads to less blood flow to the kidneys, which means less urea is delivered to the kidneys and, as a result, less urea is filtered.

What are other causative factors of prerenal azotemia?

• Shock (hypovolemic shock)

• Sudden drop in blood flow (e.g., trauma, heart conditions)

• Hemorrhage (bleeding)

• Dehydration

• Protein metabolism (e.g., high-protein diet or increased protein catabolism)

What happens in prerenal azotemia?

• The BUN/Cr ratio increases up to 20 to 30. This is due to excessive reabsorption of urea, as urea is small in size and follows water via solvent drag.

• Plasma creatinine remains normal because creatinine is larger in size than urea and is less easily reabsorbed.

What causes renal azotemia?

Renal failure, where the excretory function of the kidneys is compromised. It can be either acute or chronic.

What are some examples of conditions that cause renal azotemia?

• Glomerular nephritis

• Tubular necrosis

• Acute tubular necrosis (ATN)

• Other intrinsic renal diseases

How does renal azotemia affect BUN and creatinine levels?

Both blood BUN and blood creatinine levels are increased. However, the BUN/Cr ratio remains normal, as both are proportionally increased due to impaired kidney function.

What are some examples of conditions that cause post-renal azotemia?

• Nephrolithiasis (calculi or stones in the nephron, renal pelvis, or nephrons)

• Fluid backup, which increases urea reabsorption and causes dilatation of the ureter

• Urolithiasis (stones in the renal system, including kidneys, ureters, bladder, and urethra)

• Tumors of the bladder or prostate

• Severe infection

How does post-renal azotemia affect BUN and creatinine levels?

The BUN/Cr ratio increases, and creatinine levels also increase due to the obstruction of urine flow and impaired excretion of waste products.

What is the effect of severe liver disease on BUN levels?

The Krebs and Henselheit cycles are not effective, and BUN is not synthesized by the liver, resulting in decreased BUN levels.

How does decreased protein intake affect BUN levels?

It leads to lower BUN levels because protein metabolism produces urea, and insufficient protein intake reduces urea synthesis.

What is the impact of overhydration on BUN levels?

There is an increased volume of water in the bloodstream, which dilutes BUN levels, leading to a decrease in BUN concentration.

Why are BUN levels decreased in the third trimester of pregnancy?

There is an increase in plasma volume and fetal amino acid consumption. The fetus’s increased demand for amino acids contributes to a decrease in BUN levels.

How does Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH) affect BUN levels?

The excessive ADH (antidiuretic hormone) leads to the constant opening of Aquaporin-2 (AQP2) channels in the kidneys, causing the retention of excess water. This water retention dilutes BUN levels, leading to decreased BUN in the blood.

How can BUN methodologies be categorized?

• Enzymatic - Based on urease reactions.

• Non-enzymatic - Based on reactions such as the diacetyl or Fearon reaction, Berthelot's reaction, or Nessler’s reaction.

What are the two types of BUN methodology?

• Direct method - Requires urea.

• Indirect method - Requires NH4+ (ammonium).

What enzyme is used in the enzymatic method for BUN determination?

Urease (EC 3.5.1.5).

What happens in the urease reaction for BUN determination?

Urea reacts with water to produce ammonium ions (NH4+) and carbonate ions (CO3²⁻).

What reactions can the urease method proceed to for further BUN analysis?

Berthelot's reaction or Nessler’s reaction, both of which are indirect methods for measuring BUN.

How are ammonium ions (NH4+) measured in indirect methods?

NH4+ produced after urease action is measured using Berthelot's reaction or Nessler’s reaction. Detection is based on the differential absorbance of NADH and NAD+ at 340 nm.

What is the role of ammonium ion generation in Berthelot's and Nessler’s reactions?

The generation of ammonium ions (NH4+) is required for both Berthelot's reaction and Nessler’s reaction, as these reactions rely on the measurement of ammonium ions for determining BUN levels.

What happens in Berthelot's reaction for BUN determination?

(NH4+) reacts with phenol and hypochlorite in an alkaline medium to form indophenol blue. The chromogen is then measured at 560 nm.

How does Nessler's reaction work in BUN determination?

(NH4+) reacts with a double iodide compound (2HgI2 + 2KI), forming a yellow to orange-brown compound. The color produced is measured at 425 nm.

What is the basis of the diacetyl or Fearon reaction for BUN determination?

It is a direct method for BUN determination. It is based on the condensation of diacetyl with urea, which forms a pink chromogen called diazine.

How does the reaction with o-phthalaldehyde (OPA) and naphthylethylenediamine (NED) work in the direct method?

In this method, urea condenses with o-phthalaldehyde (OPA) and naphthylethylenediamine (NED) to form a chromogen with an absorption maximum (λmax) at 517 nm.

What is isotope-dilution mass spectrometry used for in BUN determination?

It is a definitive or reference method for measuring BUN. It is used as a highly accurate and precise technique for BUN determination.

How does isotope-dilution mass spectrometry work?

By sing an isotope-labeled internal standard (IS). The ratio of the analyte area to the internal standard (IS) area is calculated to determine the concentration of BUN.

How is the conversion factor for BUN to urea calculated?

By using the molecular weight of urea (60) and the molecular weight of nitrogen (N2) (28). To convert from blood urea nitrogen (BUN) to urea (mg/dL), multiply BUN by a factor of 2.14 (which is the ratio of the molecular weight of urea to nitrogen: 60/28).

How do you convert from urea (mg/dL) to urea nitrogen (mg/dL)?

Multiply urea by a factor of 0.46 (which is the ratio of the molecular weight of nitrogen to urea: 28/60).

What is the common practice for BUN measurement in the US and Europe?

In the US, BUN is commonly measured, while in Europe, urea is more commonly measured directly.

How do you convert from blood urea nitrogen (BUN) concentration in mg/dL to urea in mmoles/L?

Multiply BUN by 0.36 (which is 10/28, the ratio of the molecular weight of nitrogen to urea).

What specimens can be used to measure BUN?

Urea can be measured in plasma, serum, or urine.

What are some interfering substances for BUN measurement?

• Ammonium ions: These can be a source of background ammonia, which must be accounted for.

• Cigarette smoking, alcohol, and medications (e.g., barbiturates, narcotics): These can affect liver health and interfere with the test.

• Anticoagulants: High concentrations of sodium citrate and sodium fluoride must be avoided if plasma is collected, as they interfere with urease activity.

How does bacterial contamination affect BUN measurement?

It problematic because bacteria contain urease, which can lead to the bacterial decomposition of urea, affecting the accuracy of the test.

What precautions should be taken to prevent bacterial contamination in BUN measurements?

Urine should be refrigerated ASAP, and timed urine samples should be refrigerated during the collection period.

Are there any specimen requirements for BUN testing?

A fasting sample is not required for BUN testing. However, hemolyzed samples should be avoided, as erythrocytes contain 2-3 times more ammonia/ammonium than plasma concentration, which could affect the test results.

What is creatinine and how is it derived?

A waste product derived from creatine and creatine phosphate in muscle.

How is creatinine filtered in the body?

By the glomeruli (key feature), and it does not undergo significant tubular reabsorption (second key feature). Therefore, it must be removed by the kidneys as a waste product.

Where is creatinine produced in the body?

In the muscle, and its production is proportional to the muscle mass of the individual.

How is the rate of creatinine released into body fluids?

At a constant rate (third key feature of creatinine), reflecting muscle mass and its breakdown.

What causes intra-individual variation in serum creatinine levels?

The differences in muscle mass between individuals.

How is creatine synthesized?

In the liver, with the help of the kidneys, from three amino acids: Arginine, glycine, and methionine.

What is the role of Arginine:glycine amidinotransferase (AGAT)?

It is the enzyme responsible for synthesizing creatine by combining arginine (which provides guanidine), glycine (which provides acetic acid), and methionine (which provides methyl).

What is the clinical significance of creatinine in kidney function?

• Determine the sufficiency of kidney function (to assess proper kidney function).

• Determine the severity of kidney damage (for diagnosis).

• Monitor the progression of kidney disease (after diagnosis).

How is creatinine related to renal function?

Creatinine is primarily an index of renal function, as it is a waste product filtered by the kidneys and reflects how well the kidneys are functioning.

What is creatinine clearance?

It is a measure of the amount of creatinine eliminated from the blood by the kidneys, indicating how effectively the kidneys are filtering the waste.

How is estimated GFR (eGFR) calculated?

By using the Modification of Diet in Renal Disease (MDRD) equation. This equation considers 4 variables: serum creatinine, age, gender, and ethnicity. Importantly, urine creatinine values are not needed.

What should be noted about normal plasma creatinine levels?

A normal plasma creatinine level does not always indicate normal kidney function, as it can be affected by various factors such as age, race, gender, and body size.

What is the kinetic method for measuring creatinine?

Also known as the compensated Jaffe reaction, is developed to reduce the interference from conventional Jaffe reaction by accounting for interfering substances.

What are some interfering substances that the compensated Jaffe reaction reduces?

• Protein

• Glucose

• Uric acid

• Ascorbic acid

• Acetone

• Ketoacids

• Medications

How does the compensated Jaffe reaction work?

The absorbance of creatinine is measured at different time points (kinetic measurements). The absorbance at 520 nm is measured between 20 and 80 seconds, which is the time window where only creatinine reacts.

Why is the time window between 20 and 80 seconds important in the compensated Jaffe reaction?

• Acetoacetate reacts faster before 20 seconds.

• Substances like protein react slower, after 80 seconds.

• Therefore, creatinine is the main reactant during this time window, ensuring accurate measurement of creatinine without interference from other substances.

What is the enzymatic method for measuring creatinine?

• The enzymatic method for measuring creatinine involves four enzymes:

  1. Creatininase (creatinine amidohydrolase)

  2. Creatinase

  3. Sarcosine oxidase

  4. Peroxidase
     This method is preferred due to less interference, greater accuracy, and greater precision.