CITRULLINEBy

Dr.Arun Babu.N.B.II year, MD (Biochemistry)V.M.K.V. Medical College

Named after Citrullus vulgaris (Water-melon) Accumulates CIT as it’s the only way for the specific decomposition of free OH- radical.

Citrulline is a very potent scavenger of the hydroxyl radical.

CITRULLINE (CIT)

2-Amino-5-(carbamoylamino) pentanoic acid – IUPAC

Most reactive moiety in CIT – α amino group.

α carbon – oxidatively converted to aldehyde after releasing amino & carboxyl groups of CIT.

STRUCTURE

Naturally occuring basic amino acid Not a constituent of protein due to lack of codons Key intermediate in biosynthesis of Arginine & in Urea

cycle Neither present in Tissue proteins, nor in blood Present in high concn. in human brain Also present in milk. Formed in Mitochondria Leaves to Cytoplasm (further

reactions of urea cycle occur) FMN, FAD, Heme & THB – cofactors for ARGCIT.

L-CITRULLINE

Intestinal epithelium converts Glutamine Citrulline Intestinal epithelium is the only tissue that expresses an ATP

dependent glutamate reductase necessary for this conversion.

Ornithine depletion INHIBITS urea synthesis.

Ornithine Replenishment – completely dependent on source of ARGUrea synthesis in Liver – Dependent on Citrulline (gut) &

Arginine (kidney).ARG – used by many cells to produce NO.

Windmueller & Spaeth showed that there is a continuous release of CIT from small intestine circulation.

They also showed that CIT is an end-product of Glutamine metabolism

CIT – synthesized from ARG in intestine (Arginase and Ornithine Carbamoyl Transferase are present in enterocytes)

Action of 2 enzymes that catabolise CIT is very low in intestine [Arginosuccinate synthase (ASS) & Arginosuccinate lyase (ASL)]

INTER ORGAN METABOLISM

A marker of Renal FailureA marker of Intestinal FailureA Nutritional TherapeuticTreating Muscle weakness/ Fatigue/ Dementia/ Sickle

cell diseaseFor Body buildingTo increase energyTo increase athletic performanceTo decrease high BP in children after heart surgeryTo open up veins & arteries to improve blood flow

ROLES OF CITRULLINE

Kidneys-Main organs metabolizing CIT (ASS & ASL expressed along the tubules)

To establish relation between Citrullinemia & Renal insufficiency, a study was done on rats subjected to Nephrectomy(NX) [10%-90%]

Plasma CIT – Increased in mild renal failure (10 to 33% of NX)No change in Uremia 2 well known markers of No changes in Creatininemia Uremic states.

A MARKER OF RENAL FAILURE

Another study to find a marker for onset of Renal failure – to study time course of changes in aminoacidemia (Rats 36% NX, 1-21days) Uremia Peaked 24 – 48 hrs after NX Creatininemia Creatinine Clearance – Decreased concomitantly

All the above 3markers, Returned to control values in the next few daysIncreased only during the last 2 weeks

Plasma CIT concentrationIncreased 2x , 48 hrs after NXRemained at high level for the next 20 days

In humans,Plasma CIT concentration – Increased according to the

progression of renal failure.Good correlation exists between CIT & plasma concentration

of Creatinine.

Small intestine – only significant source of circulating CIT In short bowel syndrome (SBS) patients, post-absorptive

plasma CIT Valuable biomarker in the diagnosis and outcome of intestinal failure.

Plasma CIT- correlated to small bowel length & to net digestive absorption of fat

Is a marker of functional absorptive bowel length Plasma CIT has got:

High SensitivityHigh SpecificityHigh Positive Predictive Value

A MARKER OF INTESTINAL FAILURE

Another study confirmed validity of CIT as a marker of small intestinal enterocyte mass & absorption function in SBS patients.

Serum CIT levels in SBS patients correlated well with:- Remnant Small Bowel Length- Surface Area- Digestive protein absorption- Intestinal Absorption Another study showed, serum CIT levels in SBS infants

correlated linearly with:- Bowel length- Tolerance to calorie intake- Serum CIT >19μmol/L in children with SBS- associated with

development of enteral tolerance – may be a useful predictive test.

Stratification to associate citrullinemia to intestinal disease< 10 μmol/L - Diffuse total villous atrophy10 - 20 μmol/L - Proximal-only total villous atrophy20 - 30 μmol/L - Partial villous atrophy

CIT - used to quantify radiation induced epithelial loss.

All conditions characterized by an impairment of the active intestinal massHypocitrullinemia proportional to severity of diseaseARG levels

Low CIT production = Low de novo ARG synthesis.

Hence extra ARG is to be made available to patients with intestinal failure.[But ARG is taken up by liver & Arg supplementation is unsafe in some situations because of its role as NO precursor.]

Providing CIT-Better way to fill Arg pools. Hence CIT rather than Arg should be administered when intestinal function is compromised.

A NEW NUTRITIONAL THERAPEUTIC

In a study with rats (massive intestinal resection -80%): CIT supplementation – more effective than Arg

supplementation CIT content in liver-Did not vary with CIT supplementation CIT - accumulated in all other tissues CIT - passes freely through the liver ARG supplementation-Deleterious for Nitrogen balance CIT – had positive effect on the intestinal adaptation in terms

of intestinal mucosa weight & protein content CIT- aslo able to limit the body weight loss associated with

SBS. Administering an amino acid that escapes splanchnic

extraction, it’s possible to deliver a more adequate amount of nitrogen to peripheral tissues.

In a study using old malnourished rats in which an impaired response to renutrition had been proven,:

- Increased muscle protein content (+20%, p<0.05)- Stimulating protein synthesis (+90%, p<0.05)

CIT supplementation lead to dramatic improvement of:- Nitrogen balance- Protein status

INDIRECTRelated to its ability to generate ARGRelated to its ability to stimulate Insulin secretionRelated to its ability to stimulate GH secretion

DIRECTBy stimulating protein synthesis

HOW CIT STIMULATES MUSCLES TO PROTEIN SYNTHESIS?

So far, only 1 amino acid (LEUCINE) was proven to have a direct effect on muscle protein synthesis. For this action, the availability of other essential aa along with high insulinemia is necessary (i.e., post-prandialsituation).

CIT-synthesized & released by intestine mainly in case of low protein intake or post-absorptive state.

ROLE OF CITRULLINE IN CONTROL OF PROTEIN METABOLISM-A NEW

HYPOTHESIS

Working HypothesisLEU & CIT – 2 essential aa in controlling the nitrogen

balance & muscle protein composition, depending on nutritional state.

ConclusionLEU&CIT- play same function, but in different physiologic

conditionsLEU- Allows for optimal utilization of dietary aa by

stimulating post-prandial protein synthesisCIT- Allows for preservation of the muscle proteins &

maintains minimal protein synthesis in post-absorptive state.LEU- Not metabolized in liver (lack of Transaminases)With regard to Liver metabolism,

LEU is not metabolized ; CIT is not taken up.

Auto antibodies like Anti perinuclear factor (APF), Anti keratin antibodies (AKA), Anti cyclic citrullinated peptide (Anti-CCP) antibodies – Specifically associated with RA.

In patients with RA, APF & AKA specifically bind to substrates containing modified amino acid-CIT.

A peptide-based ELISA –developed using citrullinated cyclic peptide substrates for detection of anti-CCP & it may be used as a functional replacement of immunofluorescence tests used for detection of APF & AKA.

ANTI-CCP ANTIBODIES AS A MARKER OF RHEUMATOID ARTHRITIS

To detect acute & chronic renal failure To estimate the degree of renal damage As a specific marker of normal functioning of PCT As a marker of extent & severity of villous atrophy in

coeliac disease As a marker for acute cellular rejection in small intestinal

transplant recepients. As a marker of functional absorptive bowel length Anti-CCP antibody

Important serological marker for diagnosis of RAPrognostic marker for development of erosive disease

USES OF CITRULLINE

CITRULLINEMIATYPE I

Arginosuccinate Synthase def. Autosomal Recessive Lethargy progressing to coma Mental Retardation Hyperammonemia

TYPE II Defect in mitochondrial

aspartate /glutamate carrier(CTLN2)

Autosomal Recessive Neonatal Intrahepatic Cholestasis Adult- Sudden behavioral changes Hyperammonemia Stupor Coma

Clinical Features Lethargy Hypothermia Hyperammonemia Lethargy Apnea Brain Oedema Coma Seizure Vomiting Mental Retardation

Treatment Low protein diet Arginine Supplementation Breast Milk to be avoided, as

it contains Citrulline.

High Blood levels of Ammonia & CitrullineHigh Urine levels of Orotate & CitrullineCitrullinuria (1-2g/day)

1. Lippincott’s Biochemistry – 5th Edn 2. Harper’s Illustrated Biochemistry- 29th Edn3. Stryer’s Biochemistry-7th Edn4. Devlin’s Textbook of Biochemistry-7th Edn5. Lehninger’s Principles of biochemistry-5th Edn6. Harrison’s Principles of Internal Medicine-17th edn7. Christopher Moinard, Luc Cynober; “Citrulline- A new

player in the control of nitrogen homeostasis”; The Journal of Nutrition, 6th Amino acid assessment workshop.

REFERENCES