Title: To assess kidney function of Case Studies provided

Name: Erin Harrington

Date: 13/02/15 , 20/02/15 , 27/02/15

Introduction:

Proper kidney function is extremely important for an individual for many reasons. The kidney is involved in excretion, control and composition of extracellular and intracellular fluids, hormonal function and the synthesis of 1,25 hydroxy Vitamin D. Because of the role of the kidneys in the body, any malfunction is usually clinically significant and therefore it is important to assess kidney function from time to time especially in cases such as kidney donation. There are a number of tests which help determine changes in kidney function, including the determination of urea, creatinine and protein concentrations.

Urea concentration is assessed using the spectrophotometric technique. The principle of the test is based on the relationship between NADH consumption and urea concentration. In vivo urea is converted to ammonia in the presence of the enzyme urease. Ammonia is then linked with alpha ketoglutarate in the presence of glutamate dehydrogenase (GLDH). NADH is converted to NAD in this process and it is found that NADH consumption is directly proportional to urea concentration. Based on this information, a technique was created which measures this proportionality using optical density values.

A number of methods exist which estimate protein concentration in a biological sample, including the Biuret method, the Lowry method, the Bradford method, ultraviolet absorption and the Kjeldahl method. Each method is specific to certain protein samples and therefore different biological samples may be used with the different methods. However, these methods are based on colour changes which may be altered by the amino acid composition of each protein. For this reason each method may give different results for the same protein sample so protein is measured as an estimation of concentration.

The Biuret method is quick and easy and is therefore used routinely in laboratories when estimating protein concentration. This method requires the generation of a standard curve of protein standard solutions through which the test sample can be compared. The Biuret reagent causes a colour change in protein solutions which can be measured using a spectrophotometer. The intensity of colour formed is directly proportional to the protein concentration making the extrapolation of a standard curve uncomplicated in estimating an unknown sample’s protein concentration.

Creatinine assessment is based on the methodology of the Jaffe reaction. Creatinine is reacted with alkaline picrate solution to form an alkaline creatinine picrate complex which has a red colour. Although this a widely used method, it is very sensitive to noncreatinine chromogens which produce falsely elevated creatinine values. These ‘Jaffe-positive’ substances include acetoacetic acid, pyruvic acid and hydantoin. The method requires two absorbance readings to be taken to allow for adduct formations and hence, creatinine concentration can be measured. Creatinine clearance is also calculated when using the Jaffe method which is the number of mls of blood cleared of the substance per unit time. This value is used to estimate glomerular filtration rate which is an indicator of kidney function.

These tests used in conjunction can reflect kidney function and are used routinely in hospital laboratories to do so. Abnormalities in these values can indicate a number of malfunctions of the kidney as well as many diseases and injuries involving kidney function such as pre-renal Azotemia, GIT bleeding, liver disease and renal disease or failure.

Case Studies:

Patient 1: 30 year old male being assessed for his suitability as a kidney donor for his brother.

Patient 2: 35 year old male admitted to A&E with acute renal and abdominal pain. The patient is passing glossy, bloody urine.

Objectives of Practical:

To determine urea concentration of patients’ serum samples To estimate protein concentration of the patients’ urine samples using

the Biuret method To perform a creatinine clearance test in order to indicate glomerular

filtration rate (GFR) To assess the kidney function of case studies and hence, provide

diagnostic information

Results and Calculations:

Part 1: Urea Concentration

Table 1: Absorbance @ 340nm of samples at specific time intervals

Control

Standard

Patient 1

Patient 2

Abs. @ 1 min

0.050 0.003 -0.114 0.007

Abs. @ 2 min

0.030 -0.009 -0.148 -0.058

Abs. @ 3 min

0.010 -0.026 -0.155 -0.108

Abs. @ 4 min

-0.009 -0.043 -0.162 -0.104

Abs. @ 5 min

-0.030 -0.055 -0.167 -0.219

Control Absorbance = 0.050 -(-) 0.030 = 0.080/4 = 0.020Standard Absorbance = 0.003 -(-) 0.055 = 0.058/4 = 0.0145

Standard Concentration = 8.35 mmol/L

Concentration Calculation:Abs. Unknown/Abs. STD x Conc. STD = mmol/L

Control = 0.020/0.0145 x 8.35 = 11.5 mmol/LPatient 1 = [(-0.167 -(-) 0.114)÷4]/0.0145 x 8.35 = 7.63 mmol/LPatient 2 = [(0.007 -(-) 0.219)÷ 4]/0.0145 x 8.35 = 35.54 mmol/L

Note: Control Reference Range = 5.58 – 7.54 mmol/LPatient 1 True Value = 2.48 mmol/LPatient 2 True Value = 17.9 mmol/L

Part 2: Protein Concentration by the Biuret Method

Table 2: Absorbance @ 540nm of Standard solutions

Protein Standard (g/L)

Absorbance @ 540nm

0 0.00010 0.05020 0.09840 0.14860 0.238

100 0.427

Graph 1: Absorbance @ 540nm vs. Concentration (g/L) Standard Curve

See graph on sheet 1 (Calibration curve)

Table 3: Absorbance @ 540nm of Sample solutions

Sample

Absorbance @ 540nm

Control 0.171

Patient 1

0.350

Patient 2

0.287

Control Protein Concentration = 40 g/LPatient 1 Protein Concentration = 82 g/LPatient 2 Protein Concentration = 67 g/L

Note:Control Reference Range = 40 – 50 g/L

Part 3: Creatinine Clearance Test

Table 4: Absorbance @ 500nm of samples at specific time intervals

Control

Urine

Plasma

Standard

Abs. @ 30 secs

0.051 0.112

0.080 0.021

Abs. @ 3 mins 0.141 0.223

0.133 0.075

Δ Optical Density

0.09 0.111

0.053 0.054

Note:Δ Optical Density (OD) = OD2 – OD1Urine sample was diluted 1:10Standard concentration = 177 mmol/LCreatinine Concentration = Abs. Sample/Δ OD x Standard Concentration

Creatinine Concentration in samples:

Urine = 0.111/0.054 x 177 = 363.8 mmol/LxDF(10)= 3638 mmol/L True Value = 5270 mmol/L Plasma = 0.053/0.054 x 177 = 173.7 mmol/L True Value = 64 mmol/LControl = 0.09/0.054 x 177 = 295 mmol/L Reference Range = 112 – 162 mmol/L

Creatinine Clearance using True Values:

UV/P x Correction Factor for Body Surface Area/a = Creatinine Clearance

Where:U = Urine Creatinine Conc. (mmol/L) P = Plasma Creatinine Conc. (mmol/L)

V = Volume Urine Produced in ml/min a = Body Surface Area (m2)

V = 2000ml urine per 24 hours = 1.39 ml/min

5270(1.39)/64 x 1.73/1.72 = 115.123 ml/min/m2 clearance

Clearance Reference Range = 62 – 115 ml/min/m2 in males <40 = 90 – 139 ml/min/m2 in males >40

Discussion:

The objective of these practicals was to assess the kidney function of two patients using values obtained for urea concentration, protein estimation and creatinine clearance. The methodologies applied included control sample tests to validate the data.

The urea concentrations obtained from the absorbance readings in Table 1 unfortunately could not be used to assess the patients due to the control range being out of range. This showed the importance of using a control in these assays as misdiagnosis could lead to a variety of unfavourable outcomes. The control value 11.5 mmol/L was outside the range of 5.58 – 7.54 mmol/L. However, the true values were taken into account for the diagnosis of the two patients.

Patient 1, being assessed for suitability to be a kidney donor had a value of 2.48 mmol/L. The normal range for serum urea is 1.7 – 8.3 mmol/L meaning the patient had normal urea concentration indicating that his catabolism of tissue proteins is also normal. Patient 2, who had been admitted to A&E with lower back pain, had a urea concentration of 17.9 mmol/L. This concentration is very high indicating that his kidneys may not be working well or the patient is extremely dehydrated. Further tests were done in order to diagnosis the patient properly.

The Biuret test was used to estimate protein concentration in urine samples from both patients. A control was also used in this assay which gave a value of 40 g/L upon spectrophotometric analysis, data of which is noted in Table 2. The reference range for this control was 40 – 50 g/L meaning the assay was successful and values obtained could be used in the diagnosis of the patients. The normal reference range for protein in 24 hour urine is 60 – 80 g/L.

Patient 1 had a concentration of 82 g/L which is only slightly above the reference range. It was taken to be normal as small increases in protein are usually not cause for concern. So far the patient seems suitable as a kidney donor as his kidney function is good.Patient 2 had a concentration of 67 g/L which lies in the range of normal concentration. A second protein test was done on this patient however, as kidney stones were the expected cause and this time the concentration was quite low. Low protein levels coupled with high urea concentration indicated the patient may be prone to kidney stones and he was therefore tested for their presence. Kidney stones were diagnosed and the patient underwent surgery to remove them.

The creatinine clearance test was performed using the Jaffe reaction method. This test was done on Patient 1’s plasma and urine. A control sample was used, however, the value obtained, 295 mmol/L, was outside the reference range of 112 – 162 mmol/L. This may have been due to incorrect heating of the samples as the incubation was done at 50© instead of the recommended 37©. For this reason, the data obtained for the patient’s plasma and urine creatinine were discarded and the true values were used in order to determine creatinine clearance and hence conclude the suitability to kidney donation of the patient. Each sample was measured using a spectrophotomer at both 30 seconds and 3 minutes to allow for adduct formation.

The patient’s plasma creatinine value was 64 mmol/L. The reference range for normal plasma creatinine is 60 – 110 mmol/L meaning the patient had normal creatinine concentrations in his plasma. Further tests were done on his urine sample which gave a creatinine concentration of 5270 mmol/L which was calculated taking into account a dilution factor of 1:10. This again is a normal value and the patient’s creatinine clearance was calculated using these values. The reference range for normal creatinine clearance is 62 – 115 ml/min/m2 for a male under 40. The 30 year old patient had a clearance value of 115.123 ml/min/m2 which is considered normal for this age and gender. This clearance value is an indication of glomerular filtration rate (GFR) which is a marker for kidney function.

In conclusion, Patient 2 was diagnosed with kidney stones after a series of tests which indicated he was prone to their formation. The patient was admitted for surgery and the kidney stones were removed. Patient 2 had several tests done also which assessed his kidney function. His urea and protein concentrations were normal as well as his creatinine clearance. These results indicated that the patient had a functioning kidney and there was no abnormalities. According to these tests, the patient was suitable as a kidney donor for his brother and the patients were informed. The three tests performed were a good indication of kidney function and used in conjunction, a full kidney assessment could be made.