PHA 339 - Lecture #12 (Nitrogen Metabolism)

Nitrogen metabolism overview

Nitrogen metabolism involves synthesis and breakdown of nitrogen-containing biomolecules including amino acids, proteins, nucleotides, and nucleic acids

Nitrogen cycle and biologically useful nitrogen

N₂ (~78% of atmosphere) must be converted to NH₄⁺ (ammonium) through nitrogen fixation; enzyme = nitrogenase; performed by diazotrophic bacteria

Forms of inorganic nitrogen in the body

NH₃ (ammonia) and NH₄⁺ (ammonium); NH₄⁺ predominates at physiological pH; ammonia is neurotoxic

Mechanism of nitrogen transport

Amino groups are transferred through transamination reactions catalyzed by aminotransferases using pyridoxal phosphate (PLP)

Key amino acids in nitrogen metabolism

Alanine transports nitrogen from muscle to liver; Glutamine transports ammonia in blood; Glutamate is the primary amino group carrier; Aspartate provides nitrogen to the urea cycle

Nitrogen disposal and waste products

Three nitrogenous waste products are urea (amino acid and pyrimidine catabolism), creatinine (creatine phosphate metabolism in muscle), and uric acid (purine catabolism)

Urea cycle overview

The urea cycle converts toxic ammonia to urea in hepatocytes; nitrogen enters as ammonium or aspartate; urea is water-soluble and excreted by kidneys

Creatinine metabolism and anabolism

Arginine and glycine form guanidinoacetate, which is methylated to form creatine

• In muscle, creatine is phosphorylated to form phosphocreatine

• Phosphocreatine is used as an energy source to regenerate ATP

• Creatinine is formed spontaneously (non-enzymatically) as a byproduct and is renally

cleared through glomerular filtration

Nitrogen balance

Nitrogen balance = nitrogen intake − nitrogen excretion; intake mainly dietary protein; excretion mainly urea in urine

Positive vs negative nitrogen balance

Positive = growth, pregnancy, tissue repair; Negative = starvation, trauma, severe illness

Role of gut bacteria in nitrogen balance

Gut bacteria recycle nitrogen; urease converts urea → ammonia + CO₂

Protein turnover

Continuous synthesis and degradation of proteins maintains amino acid pool; half-life = time required for 50% protein degradation

Protein degradation pathways

Ubiquitin-proteasome pathway (selective) and lysosomal degradation pathway (non-selective)

Origin of amino acids in the body

Amino acids originate from dietary protein digestion and synthesis of nonessential amino acids

Essential vs nonessential amino acids

9 essential amino acids must be obtained from diet; 11 nonessential amino acids can be synthesized

Conditionally essential amino acids

Normally synthesized but required from diet during illness, growth, or metabolic stress

Dietary protein digestion

Proteolytic enzymes digest proteins; enzymes secreted as zymogens; stomach HCl denatures proteins and activates pepsinogen → pepsin

Pancreatic digestion and absorption

Pancreas secretes bicarbonate to raise intestinal pH; proteases include trypsin, chymotrypsin, elastase; amino acids absorbed in small intestine → portal vein → liver

Synthesis of nonessential amino acids

Carbon skeleton from glycolysis or TCA intermediates; nitrogen added via transamination; cysteine sulfur from methionine; tyrosine synthesized from phenylalanine

Amino acid catabolism

Carbon backbone used for energy; nitrogen removed and converted to urea

Glucogenic vs ketogenic amino acids

Glucogenic → glucose via gluconeogenesis; Ketogenic → ketone bodies (lysine, leucine); Both = isoleucine, phenylalanine, threonine, tryptophan, tyrosine

Nitrogen disposal pathway

Amino group transferred to α-ketoglutarate forming glutamate; glutamate releases ammonium or transfers nitrogen to aspartate

Glutamate dehydrogenase reaction

Glutamate → α-ketoglutarate + NH₄⁺ via oxidative deamination

ALT and AST enzymes

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are transaminases used clinically as markers of liver damage

Clinical interpretation of ALT and AST

Elevated levels indicate hepatocyte damage; AST:ALT >2 suggests alcoholic liver disease

Hyperammonemia

Elevated blood ammonia due to impaired nitrogen disposal

Hyperammonemia causes

Liver disease, kidney failure, urea cycle defects

Hyperammonemia symptoms

Confusion, lethargy, seizures, vomiting, coma

Phenylketonuria (PKU)

PKU results from deficiency of phenylalanine hydroxylase causing increased phenylalanine and decreased tyrosine

Phenylalanine hydroxylase function

Converts phenylalanine to tyrosine

Neurologic damage in PKU

High phenylalanine disrupts amino acid transport into the brain

PKU dietary restriction

Avoid aspartame because it is metabolized into phenylalanine

Nucleotide structure

Nucleotide = nitrogenous base + pentose sugar + phosphate group

Origin of nucleotides

Nucleotides are primarily synthesized or recycled; minimal dietary contribution

Purines vs pyrimidines

Purines = adenine, guanine; Pyrimidines = cytosine, thymine, uracil

De novo nucleotide synthesis

Synthesis of nucleotides from small precursor molecules

Salvage pathway

Recycling of nitrogenous bases from nucleotide breakdown

Purine synthesis pathway

Ribose-5-phosphate → PRPP → IMP → AMP or GMP

Pyrimidine synthesis pathway

Glutamine + CO₂ → orotic acid → UMP

Ribonucleotide reductase

Converts ribonucleotides to deoxyribonucleotides

Thymidylate synthase

Converts dUMP to dTMP required for DNA synthesis

Purine catabolism

Purines degraded to uric acid

Xanthine oxidase enzyme

Catalyzes formation of uric acid during purine breakdown

Uricase enzyme

Converts uric acid to allantoin in other mammals; humans lack uricase

Pyrimidine catabolism products

β-alanine, β-aminoisobutyrate, NH₄⁺, CO₂

Gout overview

Gout is caused by elevated uric acid leading to precipitation of monosodium urate crystals

Symptoms of gout

Painful inflammatory arthritis; crystals often deposit in joints; may also cause kidney stones or nephropathy

Causes of gout

Overproduction of uric acid or reduced renal excretion

Factors increasing uric acid production

PRPP synthetase overactivity, tumor lysis syndrome from chemotherapy, high purine diet

Antimetabolites

Drugs that interfere with nucleotide synthesis by mimicking substrates

Gemcitabine example

Gemcitabine is a cytidine analog incorporated into DNA causing chain termination and apoptosis

Cells affected by antimetabolites

Rapidly dividing cells including cancer cells, bone marrow, hair follicles, and GI tract

Which of the following is TRUE regarding the nitrogen cycle?

NH₄⁺ is used to build amino acids

Which of the following best describes how nitrogen is transported from peripheral tissues to the liver through the bloodstream?

As amino groups on alanine or glutamine

Which of the following are TRUE for nitrogen disposal? Select all that apply

Urea is the most abundant nitrogenous waste product; Urea is a byproduct of amino acid catabolism

Which of the following are TRUE regarding amino acid metabolism? Select all that apply

Excess amino acids can be converted to glucose; Amino acids can be catabolized as an energy source

How are dietary proteins absorbed into the bloodstream?

As amino acids

Leucine is a ketogenic amino acid. This means that

Its carbon backbone is broken down to produce ketone bodies

Which of the following best describes the urea cycle?

Converts ammonia to the less toxic urea

Why do elevations of ALT and AST occur in liver injury?

Due to cell damage resulting in release of intracellular enzymes

A 14-year-old girl with PKU experiences a seizure after consuming a sweetener. What caused it?

Ingestion of aspartame because it contains phenylalanine

Inosine monophosphate (IMP) serves as a precursor for

GMP and AMP

Which of the following are TRUE in pyrimidine catabolism? Select all that apply

The pyrimidine ring is broken down; Ammonia is produced

A drug that inhibits thymidylate synthase interferes with cancer cell proliferation by

Inhibiting DNA synthesis by altering pyrimidine production

Ammonium to ammonia ratio at physiological pH

Approximately 100:1 (NH4+ : NH3)

Why nitrogen cannot be transported as free ammonia

Free ammonia is toxic (neurotoxic)

Preferred nutritional energy sources before amino acids

Glucose and triglycerides

What happens to excess amino acids

Converted to other molecules such as glucose because amino acids are not stored

Which organ is the primary site of de novo nucleotide synthesis

Liver

Why humans are prone to uric acid accumulation

Humans lack the enzyme uricase

Urea

Byproduct of amino acid and pyrimidine catabolism; produced in the liver via the urea cycle

Nitrogen

transported as the amino acids alanine and glutamine

This enzyme is produced by bacteria and breaks down urea

urease

This enzyme transfers an amino group from an amino acid to an alpha-keto acid

transaminase / aminotransferase

This enzyme converts ribonucleotides to deoxyribonucleotides

ribonucleotide reductase

This enzyme produces uric acid

xanthine oxidase

This enzyme removes ammonium from glutamate through oxidative deamination

glutamate dehydrogenase

This amino acid is the primary amino donor in transamination reactions

glutamate

This amino acid is synthesized by hydroxylation of the essential amino acid phenylalanine

tyrosine

These two amino acids are primarily responsible for transporting nitrogen through the bloodstream

alanine and glutamine

This amino acid enters the urea cycle directly

aspartate

This amino acid releases ammonium which enters the urea cycle

glutamate

Uracil is a precursor for this nitrogenous base found only in DNA

thymine

This is the name for the pathway that detoxifies ammonium

urea cycle

This is the name for the pathway that recycles nitrogenous bases

salvage pathway

This pathway is responsible for non-selective degradation of proteins into amino acids

lysosome pathway

This is the name for the pathway that synthesizes nitrogenous bases

de novo pathway

This is the most abundant nitrogenous waste product

urea

This nitrogenous waste product is produced in muscle cells

creatinine

This nitrogenous waste product is produced by purine catabolism

uric acid

This is the final nitrogenous waste product formed by amino acid catabolism

urea

The enzyme uricase forms this nitrogenous waste product in non-human animals

allantoin

Patients with hepatic encephalopathy experience neurological dysfunction due to the elevation of this substance

ammonia / ammonium

A patient with gout should limit their dietary intake of this type of nitrogenous base

purines

Blood levels of this will be elevated in patients who have a genetic defect in the urea cycle

ammonia / ammonium

In alcoholic liver disease, this liver enzyme is predominately elevated

AST

This category of drugs used in cancer treatment interferes with nucleotide anabolism

antimetabolites

A new drug for the treatment of hypertension is developed by an academic research group. When the drug is tested in mice, the drug increases blood levels of uric acid as a side effect. When the drug is tested in humans, there is no effect on uric acid levels. This drug is most likely an inhibitor of which enzyme?

uricase

Which of the following forms of nitrogen is used to build amino acids?

NH4+

Which of the following is TRUE for transamination? Select all that apply.

the amino group of an amino acid is transferred to an alpha-keto acid; one of the products formed is an amino acid