*Jamal S Al Wakeel M.D., *Durdana Hammad M.D., *Abdul Karaem Al Suwaida M.D., *Nauman Tarif M.D., ♦ AbdulRauf Chaudhary, ♦ Arthur Isnani M.D., ♠ Waleed Ahmed Albedaiwi M.D., *Ahmad H Mitwalli M.D. ●Shaik Shaffi Ahmad PhD. Division of Nephrology, Department of Medicine, ●Department of community medicine, ♦ Research centre, King Khalid Hospital, King Saud University. ♠ King Fahad National Guard Hospital Riyadh, KSA.

Glomerular Filtration Rate (GFR) is a surrogate marker of kidney function Monitoring of GFR help delineate kidney function. The methods for estimation of GFR are not complete proof and have some limitations.

Serum Creatinine and Creatinine clearance used traditionally poorly detect early GFR declines Further serum creatinine is affected by muscle, mass, diet and inflammation etc. Creatinine clearance is affected by serum creatinine and affected by urine collection method Inulin clearance is gold standard, it complexity procedure, not suitable routinely. EdTA and DTDA have radioactive exposure, couscous handling, and high cost have limited the use Serum cystatin C and non radio-labeled cold Iothalamate are new promising markers --lengthy laboratory procedures.

Prediction equations provide a rapid method of assessing GFR. GFR prediction equations like Cockcroft-Gault and Modification of Diet In Renal Disease (MDRD) are recommended by K-DOQI Guidelines for estimation of GFR.

Frossart M et al. (J Am Soc Nephrol 2005 Mar; 16(3): ) Caucasian adults Cockcroft-Gault Formula was less precise than MDRD. Zuo L et al. (Am J Kid Dis 2005: 45(3)463-72) found MDRD equations needs modification for estimating GFR in Chinese patients Mahajan S et al. (J Nephrol 2005; 18 (3) ) MDRD 1 and MDRD 2 equations were most precise and MDRD 1 prediction equation for GFR was the most accurate in measuring the GFR in Indian population Aizawa M in 2006 (Nippon 2006;48(2) 62-66). Study was conducted in 100 patients concluded that Cockcroft-Gault formula gave highest correlation with GFR in Japanese patients with CKD

Further Van Deventes HE et al (Clin Chem 2008 ; 54(7): ) used plasma clearance of chromium-51 EdTA to measure GFR and compared with MDRD measured GFR in black South Africans. He concluded that MDRD 4-V equation to be most suitable for measurement of GFR in black South Africans.

Objective: The aim of the study was to compare the various predictive equations, MDRD, Cockcroft-Gault, and other markers of GFR Reciprocal Cystatin C and Reciprocal creatinine for the measurement of GFR Compare them with the Gold Standard Inulin clearance. To find out the most applicable one for Saudi Population.

Method: It was a cross sectional study. Approved and funded by King Abdulaziz City Of Science and Technology (KACST) and King Saud University, Riyadh, Saudi Arabia. Conducted from January 2005 to June 2007 at King Khalid University Hospital King Saud University Riyadh Saudi Arabia.

Inclusion criteria : Patients older than 18 yrs with the diagnosis of CKD Post-kidney transplant patients stable for three months, Healthy subjects without any renal disease and not on any medication. Exclusion criteria : Patients with acute renal failure, edema, or heart failure, ascitis, pregnant ladies or patients with infection.

Data collected included: Gender, age, weight, height, body surface area (B.S.A.), and blood pressure (B.P.) Blood samples were taken for estimation of serum cystatin C, serum creatinine simultaneously during Inulin clearance test Serum Cystatin C was measured by Nephelo- immunoassay Serum Creatinine was analyzed by Jaffe’s method in the central laboratories at KKUH on third generation automated clinical chemistry Dimension RxL analyzer (Dade Behring Inc, Germany)

GFR was calculated using following prediction equations: Cockcroft-Gault (CG) equation : GFR = ((140 – Age (years) x Weight (kg) x 1.23/serum creatinine (umols) FOR MALES GFR = ((140 – age (yrs) x Weight (kg) x 1.02 /serum creatinine (umols) FOR FEMALES MDRD : GFR = 1.86 x (Scr) x (age) x (0.742 if patient is female) GFR was corrected for Body Surface Area as follows: Corrected GFR =(uncorrected GFR x 1.73)/Body Surface Area

Measurement of GFR by Inulin Clearance Test: Informed Consent was taken from volunteers. Patients fasted over night. Two I.V. Canulae were placed in both arms one for the blood sample extraction and the other for Inulin injection and infusion. Urine and Blood sample were taken at zero time for biochemical analysis. Loading close of Calculated Inuline was given exactly according to manufacturers instructions. Where loading dose = (250 mg/L x 15% of the total body weight (TBW). While continuous infusion dose rate = Required Plasma Conc. X Estimated GFR by Cockroft- Gault Formula ml/min

Loading dose was followed by Constant infusion of Inulin at Calculated Rate. Oral hydration of 100 ml/hr was maintained Blood samples were drawn from the arm opposite to the infusion site After 60 minutes of infusion first urine or blood sample was drawn. Then after 30 minutes until 5 hours.Samples were processed for Inulin Estimation Inulin will be estimated according to the Standard Method described by Previous Workers (RJ Davidson and Sackness) GFR will be measured as: GFR in= Uin x V Pin Where Uin and Pin are Inulin concentration in the urine and Plasma and V is the urine flow rate ml/min.

Statistical analysis: Quantitative variables such as age, height, weight, BSA, BMI, serum creatinine, and GFR were presented as mean + standard deviation. Statistical analysis was carried out using SPSS 11.5 for Windows. The difference of GFR calculated by Cockcroft-Gault and MDRD was compared to Inulin clearance using Bland and Altman plots and Pearson’s correlation Mann Whitney U- test. The P value <0.05 was considered as significant.

Results: 32 Saudis consented for the procedure. 15 CKD patients, 9 Post-Kidney Transplant patients, 8 healthy subjects. There were 19 males (59.37%) and 13 (40.6%) females The mean age of all the patients was 42.3 ± 15.2 years (19 – 74 years).

A linear relationship was found between GFR determined by MDRD and GFR determined by Inulin (y=0.9706x ), R 2 = R 2 = R 2 =0.9073

Limitations of present study Number of patients is small Number of old patients are small However our results are consistent and statically significant and thus acceptable.

All the markers of GFR, MDRD prediction equation highly correlated to GFR-Inulin than others While Cockcroft-Gault Formula was the next Serum Cystatin C, Serum Creatinine, Reciprocal of cystatin C and Reciprocal of serum Creatinine was inferior to MDRD or Cockcroft-Gault prediction equation. MDRD equation to be the best suitable and valid prediction equation for the measurement of GFR in Saudi population In health and also in various clinical presentations like renal transplant patients and CKD patients CONCLUSION

My special thanks to…. Contributors on the study: Dr. Fathia Sulimani Dr. Abdo Qudsi Dr. Iqbal Shah Mohammed Naeem (Research Assistant) 1.Salah Haddeen (Research Assistant) 2.Hamsa Veni Wilson (Head Nurse, PDDC) Funded and Supported By King AbdulAziz City Of Science And Technology. (KACST) and KING SAUD UNIVERSITY.

King Khalid University-King Saud University 1.Dr. Fathia Sulimani( 2.Dr. Mohammed Al Ghailani 3.Dr. Iqbal Shah 4.Dr. Shahid Qayyum 5.Dr. Abdo Qudsi 6.Dr. Habib Ur Rahman 7.Dr. Salman Imtiaz 1.Hamsa Veni Wilson (Head Nurse, PDDC) King Khalid University-King Saud University

Further the estimation equations are not validated for the following conditions Individuals with high normal renal function Pregnant ladies Children Unusual body mass Malnourished pr morbid obesity Certain ethnic groups

Further Bland and Altman plots demonstrated that MDRD calculated GFR was most accurate when compared with gold standard Inulin clearance. Fig 1, 2.

Correlation coefficient for other markers were: Cockroft-Gault r =0.95 Cystatin C r = Serum Creatinine r = (inferior to those obtained between GFR from predicted equations) MDRD r= 0.97 Inulin clearance Reciprocal of Cystatin C and Serum Creatinine also showed correlation with Inulin GFR but 0f lesser magnitude than prediction equations Further to test the versatility of predictive efficacy of equations for calculation of GFR, calculation of GFR were done in different groups of the patients as follows.

A. Across gender Among Males: There were 19 (59.37%) males Mean age was 41.89±16.7 years ( 19 – 74yrs). Height was ±7.6 cm. Weight was 69.34±18 kg Body Surface Area (BSA) was 1.77± Serum Creatinine was ± µmol/L. GFR Inulin was 58.45±32.76 ml/min. GFR Cockroft-Gault was ±32.48 ml/min The GFR MDRD was 56.45±33.56.

The difference between GFR Cockcroft-Gault was 1.037±9 vs. Inulin (GFR Inulin – GFR Cockcroft-Gault) While the difference between the GFR vs. Inulin (GFR Inulin – GFR MDRD) was only 1.09 ± 7 ml/min. In comparison to the Cockcroft-Gault GFR, the GFR MDRD was more close to Inulin GFR. The correlation coefficient was r = 0.977, p=0.000 for GFR calculated by MDRD vs. inulin clearance While r = p=0.000 is for GFR Cockcroft-Gault vs. Inulin clearance.

Correlation coefficient for Cystatin C was r= (p=0.001) and Serum Creatinine was r= (p=0.01) and the correlation coefficient between the reciprocal of serum cystatin C vs. GFR Inulin was r=0.822

Among Females: There were 13 females Mean age 42.92±13.2 years Height was 151.7±7.4 cm Weight was 67.6±17.9 kg, BSA was 1.67±0.21; Serum Creatinine was 203.7±149.8 µmol/L. GFR Inulin was 39.78±32 ml/min while the GFR Cockcroft- Gault was over estimated was 51.69±35 ml/min and GFR MDRD was 47.38±32.

In comparison to the Cockcroft-Gault estimated GFR, the GFR calculated by MDRD was more close to Inulin GFR. The correlation coefficient was r = (p=0.000) for GFR calculated by MDRD vs. inulin clearance While r = (p=0.000) was for GFR calculated by Cockroft- Gault vs. Inulin clearance. The correlation coefficient between the serum cystatin C vs. GFR Inulin was r=0.84 (P=0.001) while for serum creatinine vs. GFR Inulin was r=0.72 (p = 0.01)

GFR across Age Age less than 40 years: Twelve (37.5%) patients. Mean age was 26.9±6.9yrs (19-39 years). Mean height was 162.5±9.9 cm Mean weight was 70.2±25.9 kg and BSA was 1.75 ±0.31. Serum Creatinine was 179.8±149.1 µmol/l Inulin-GFR was 60.5±35 ml/min GFR calculated by Cockcroft-Gault was 69.39±33.8 ml/min and GFR calculated by MDRD was 67.5±31.8 ml/min. GFR Inulin – GFR Cockcroft-Gault was while the difference GFR Inulin – GFR MDRD was only -0.98ml/min. GFR MDRD correlated best with the inulin clearance r= (p=0.0001) while correlation coefficient for Cockcroft-Gault- GFR vs. Inulin was r=0.935 (p=0.0001). Correlation coefficient for cystatin C was 0.731, and for serum creatinine was r=0.763in relation to inulin clearance. MDRD GFR was closest to GFR measured by Inulin

Age 40 – 60 years: Total 15 patients (46.8%) were between 40 – 60 years of age. Inulin estimated GFR was 46.9±34.6 ml/min. Cockcroft-Gault equation overestimated the GFR was 51.27±34.17 ml/min. The GFR estimated by MDRD was closer to Inulin measured GFR being 45.8±34.4 ml/min. The difference between GFR calculated by Cockcroft-Gault was -4.37±8.1 vs. Inulin while GFR Inulin – GFR MDRD was only 1.1±5.7 ml/min. GFR calculated by MDRD was more close to Inulin GFR r = (p=0.000) while for Cockcroft- Gault vs. inulin was r = 0.97 (p=0.000) Correlation coefficient for Cystatin C r= and Serum Creatinine was r=0.651 Age more than 60 years: MDRD was more correlated to GFR-Inulin (r = 0.984, p = 0.001) than Cockcroft-Gault-GFR (0.938, p = 0.01).

C. Across various groups of patients. A. Healthy subjects: Eight (8) healthy patients, 4 males (50 %) and 4 females (50%) The Inulin-GFR was ± 28.61, GFR-Cockcroft-Gault was 94.5 ± indicating statistically significant overestimation of GFR (p = 0.05) The GFR-MDRD was ± 27 ml/min MDRD more superior to Cockcroft-Gault (r = 0.934, p < ) vs. (r = 0.863, p=<0.0001), respectively.

B. GFR across BMI: Patients with BMI less than 30 kg/m2 There were 23 patients with BMI less than 30 kg/m2 The mean age was (19-74 yrs), mean height of ± 8.1 cm (150 – 176 cm), mean weight of ± 10.6 kg (42.6 – 91 kg), Mean Body surface area of 1.67 ± Serum creatinine of ± (51 – 815 µmol/L) The overall mean value of GFR-Inulin was 52.6 ± 35.16ml/min (9 – ml/min). GFR-Cockcroft-Gault was 55.4 ± and the GFR-MDRD was 52.5 ± 36.4 ml/min. Both equations well correlated with Inulin Clearance with MDRD more superior to Cockcroft-Gault (r = 0.987, p < ) vs. (r = 0.971, p = < 0.001), respectively.

BMI More Than 30 kg/m2: There were 8 patients with BMI >30 kg/m2. The mean age was yrs (29-70 yrs) Mean height of ± 13.6 cm ( cm) Mean weight of 85.5 ± 24.3 kg (62.4 – kg) Mean Body surface area of 1.9 ± 0.33 and serum creatinine of ± (73.5 – 591 µmol/L) was studied. The overall mean value of GFR-Inulin was 47.8 ± 29.6 ml/min (5.9 –92 ml/min). GFR-Cockcroft-Gault was and the GFR-MDRD was 51 ± 23.2 ml/min. MDRD better correlated with Inulin Clearance (r = 0.989, p < ) than GFR-Cockcroft-Gault (r = 0.976, p = < 0.001).

Post-Renal Transplant Group Total 9 patients with mean age of years (20 – 64 years) Mean height of ± 10.2 cm (144 – 1786 cm) Mean weight of 65.9 ± 9.1 kg (53.4 – 80 kg) Mean Body surface area of 1.7 ±.008 Serum creatinine of ± 43.4 (88 – 195µmol/L). GFR-Inulin was 62.5 ± ml/min. GFR-Cockcroft-Gault was ± (p = > 0.05) while GFR-MDRD was ± (p> 0.05). Both equations well correlated with GFR-Inulin. However, GFR-MDRD showed better correlation with GFR- Inulin (r=0.946, p <0.0001) as compared to GFR-Cockcroft- Gault (r=0.929, p< ).

Chronic Kidney Disease patients: Chronic Kidney Disease Patients Fifteen CKD patients with mean age of 46.4 ± 16.6 years (19 – 74 years) Mean height of 162 ± 9.9 cm (144 – 178 cm) Mean weight of 74.0 ± 22.3 kg (50.6 – kg) Mean Body surface area of 1.81 ± 0.28 Serum creatinine of ± (127 – µmol/L) GFR-Inulin was 25.8 ± ml/min The mean GFR-Cockcroft-Gault was 30.9 ± 18.3 ml/min. GFR-MDRD was 25.1 ± 13.3 ml/min. Both equations well correlated to GFR-Inulin with relative more advantage for MDRD compared to Cockcroft-Gault equation (r=0.943, p < vs. r=0.923, p<0.0001) respectively.

Across variable GFR Patients with GFR less than 30 ml/min Eleven patients (34.3%) Mean age in this group was 44.6±17.5 years Height was ±10.2 cm Weight was 71.11±17.9 kg Mean BSA was 1.76±0.22 Mean serum creatinine was 352±194.5 µmol/L. GFR-Inulin was 19.9±7.5ml/min. GFR-Cockcroft-Gault over-calculated GFR (24.1±8.4ml/min) while GFR-MDRD was 19.2±8.5 ml/min. Significant correlations were shown between GFR-Inulin vs. Cockcroft-Gault (r=0.715, p=0.013) and GFR-Inulin vs. GFR-MDRD (r=0.882, p=0,001) with more advantage to MDRD equation.

Patients with GFR more than 60 ml/min: Eleven patients (34.3%) with GFR more than 60 ml/min Mean age years Height of cm BSA of serum creatinine of µmol The Inulin measured GFR was ±17.4ml/min. The Cockcroft-Gault calculated GFR which was /min while MDRD calculated GFR was ml/min. The correlation coefficient between GFR calculated by Cockcroft-Gault formula vs. Inulin measured GFR was r = 0.85, p = while correlation coefficient r = p=0.000 for GFR calculated by MDRD.

Patients with GFR more than 90 ml/min: Five patients (15.6%) with GFR more than 90 ml/min Mean age Height of cm BSA and serum creatinine of µmol/L were studied. The Inulin measured GFR was ±15.5ml/min. The Cockcroft-Gault calculated GFR which was ml/min while MDRD calculated GFR was ml/min, The correlation coefficient between GFR calculated by Cockcroft-Gault formula vs. Inulin measured GFR was r = 0.823, p = 0.08 while correlation coefficient r = 0.88 p=0.05 for GFR calculated by MDRD.