Cystatin c

CYSTATIN-C

Presented byDr.Brijesh Mukherjee

P.G.,Biochemistry.

HISTORY

• It was first described as “gamma trace in 1961 as a trace protein together with other ones(such as beta trace) in CSF and in urine of patient with renal failure.

• Grubb and Lofberg first reported its amino acid sequencence

• They noticed it was increased in patient of CRF• It was first proposed as a measure of GFR by

Grubb and co-workers in 1985

MOLECULAR BIOLOGY

• Cystatin superfamilly encompasses protein that contain multiple cystatin like sequences

• Some members are active cysteine proteinase inhibitors

• There are 3 inhibitory families in the superfamily-• 1) Type 1 cystatins(stefins)• 2) Type 2 cystatins• 3)Kininogens• Type 2 are a class of cysteine proteinase inhibitors

found in human fluids and are protective in function

MOLECULAR BIOLOGY(CONT.)

• They are located in short arm of chromosome 20 which contain majority of type 2 cystatin genes and pseudogenes

• Highest level found in semen followed by breast milk,tears and saliva

SOME FACTS ABOUT CYSTATIN-C• Cystatin-c is a non glycosylated basic protein(isoelectric pH 9.3)• It has a crystal stucture characterized by short alpha helix and a long

alpha helix running across a large antiparallel 5 stranded beta sheet• It has 2 disulfide bonds• 50% of the molecule carry a hydroxylated proline• It forms two dimers(molecule pairs)• In human all cells with nucleus produce cystatin c as a chain of 120

amino acids• It is a potent inhibitor of lysosomal proteinases• It is also an important inhibitor of extracellular cysteine proteases• It has a low molecular weight of 13.3 kilodaltons•

SRUCTURE OF CYSTATIN C

DIMERSIn biochemistry a dimer is a macromolecular complex formed by two, usually covalently

bound, macromolecules like proteins or nucleic acids. It is a quaternary structure of a protein.

ROLE IN MEDICINE

• KIDNEY FUNCTION• It is removed from bloodstream by

glomerular filtration by kidneys• if the function of kidneys decrease and GFR

falls, level of cystatin-c in blood increases• So it has been suggested that cystatin-c

might predict the development of CRF

ROLE IN MEDICINE (CONT.)• Levels of cystatin-c are altered in following conditions• 1)Cancer patient• 2)Thyroid dysfunction• 3)Glucocorticoid therapy• 4)Cigarette smoking• 5)HIV infection• 6)Increased levels in MI,stroke,heart failure,peripheral arterial

syndrome• 7)Increased in metabolic syndrome• 8)Increased in alzheimers disease• 9)Levels decreased in atheroslerosis and aneurysmal(saccular

bulging) lesions of aorta

STAGES OF KIDNEY DISEASENORMAL Healthy kidney GFR >90 ml/min

STAGE 1 Kidney damage with normal/elevated GFR

GFR >90 ml/min

STAGE 2 Kidney damage with mild decrease in GFR

GFR 60-89 ml/min

STAGE 3 Kidney damage with moderate decrease in GFR

GFR 30-59 ml/min

STAGE 4 Kidney damage with severe decrease in GFR

GFR 16-29 ml/min

STAGE 5 Kidney failure(end stage renal disease,ESRD)

GFR <15 ml/min

PREVALANCE

METABOLIC EFFECTS IN CRDStage (Renal dysfunction) GFR

(ml/min/1.73 m2)

Metabolic Consequences

Normal renal function – Persons at increased risk or with early renal damage

>90  

Mild renal insufficiency (ERI) 60-89* PTH levels start to rise (GFR ~ 60-80)

Moderate renal insufficiency (CRI) 30-59 Decrease in Calcium absorption (GFR <50)

Lipoprotein activity falls.

Malnutrition.

Onset of LVH.

Severe renal insufficiency (Pre-ESRD) 15-29 Triglyceride levels start to rise.

Onset of Anemia (EPO deficiency).

Hyperphosphatemia.

Metabolic acidosis.

Hyperkalemia tendency.

ESRD (Uremia) <15 Azotemia develops.

PROGRESSION OF C.R.D.

CRITERIA FOR C.R.D.

G.F.R.

• GFR is a measure of rate at which water and dissolved substances(low molecular weight ultrafiltrable compounds) are filtered out of blood per unit time through kidneys

• NORMAL GFR• Males-150 ml/min• Females-130 ml/min• Serum creatinine is most common marker used

to measure GFR

LIMITATIONS OF CREATININE AS A MARKER OF GFR

• NON RENAL FACTORS-• 1.Gender• 2.Ethinicity• 3.Diet• 4.Muscle mass• 5.Drugs affecting tubular secretion of creatinine• CLINICAL FACTOR-• Poor sensitivity for CKD- Creatinine blind range• Creatinine remains normal until 50% renal function is lost• Insensitive to loss of GFR in Stage-2 and Stage-3 in CKD• ANALYTICAL FACTOR-• Non specific bias frequently reported with Jaffe Assay Method

ADVANTAGE OF CYSTATIN-C AS GFR MARKER

ADVANTAGE COMMENT

Virtually unaffected by non renal factors Muscle mass/weight/height,age(>1 year)-cystatin-c parallels age related decrease in GFR and can be used in children

Sensitive to so called creatinine blind range Enables early detection and treatment of CKD

Can be used to detect and monitor kidney diseases in patient with hepatic diseases

Creatinine for GFR in liver disease not recommended

Correlates to appearance of microalbuminuria Clinical studies suggest that very early renal failure may be the first clinical indication of progressive renal damage associated with diabetis

CONTRAINDICATION OF CYSTATIN-C ESTIMATION

• THYROID FUNCTION • Levels of cystatin-c are sensitive to change in

thyroid function and should not be performed without knowledge of patients thyroid status

• CORTICOSTEROIDS• Cystatin-c concentrations are affected in

patients of impaired renal function receiving corticosteroids

LABORATORY MEASUREMENT• ASSAY PRINCIPLE-• Cystatin-c in the sample binds to the specific anticystatin-c antibody which is

coated on latex particles and causes agglutination• The degree of turbidity caused by agglutination is measured optically and is

proportional to the amount of cystatin-c in the sample by a method called TURBIDIMETRY.

• REFERENCE VALUE-• Males-0.52-0.98 mg/dl• Female-0.52-0.90 mg/dl• Normal value decreases until first year of life,then remains stable before

increasing after age of 50 years• NOTE-• Cystatin-c can be measured from a random sample of blood from which RBC

and clotting factors have been removed(i.e. serum)

TURBIDIMETRY

• Some analytical methods give an insoluble product in finely divided form so that the particles remain in suspension

• If a beam of light passes through,some of it is scattered-TYNDALL EFFECT

• Turbidimetry measures the reduction of intensity of the incident beam and is similar to the study of light absorption in spectrophotometry

• Turbidimetric measurements are done with usual types of photometers

TURBIDIMETRY COMPONENTS

TURBIDIMETRY AND NEPHELOMETRY

TURBIDIMETER

STANDARD GRAPH OF TURBIDIMETRY

% INCREASE IN CYSTATIN-C LEVELS AFTER 50 YEARS

SENTITIVITY AND SPECIFICITY OF CYSTATIN-C/CREATININE

GRAPH OF CYSTATIN-C(STD.)

GRAPH OF CYSTATIN C ASSAY

CYSTATIN-C CONC. VS OPTICAL DENSITY GRAPH

GENTIAN CYSTATIN-C GFR CALCULATOR

• The calculator gives instant conversion of cystatin-c measurement to standard GFR units

GFR CALCULATORCYSTATIN-C(mg/dl) GFR(ml/min)0.5 2170.6 1670.7 1330.8 1100.9 931.0 801.1 701.2 611.3 551.4 491.5 451.6 411.7 371.8 34

1.9 32

2.0 29

3.0 16

3.5 13

4.0 11

4.5 09

5.0 08

5.5 07

6.0 06

6.5 05

7.0 05

7.5 04

CONCLUSION

• The prevalence of chronic renal diseases are increasing in INDIA especially with an increase in cases of diabetis

• Hence it is important to diagnose renal disease at an early stage to decrease morbidity and mortality

• The use of CYSTATIN-C as a marker of CRD can be an important tool to monitor renal disease.

THANK YOU