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Research Paper
Volume : 3 | Issue : 9 | September 2014 • ISSN No 2277 - 8179
Detection of Microalbuminuria by
Measuring Urinary Protein To Creatinine
Ratio Type 2 Diabetes Mellitus Patients.
BHAVANI K
Medical Science
KEYWORDS : Microalbuminuria, Protein
creatinine ratio, type 2 diabetes mellitus
9th Term MBBS Student, M S Ramaiah Medical College and Hospitals
* DR. VANITHA GOWDA Associate Professor, Dept of Biochemistry, M S Ramaiah Medical College, Bangalore –
560054. India *Corresponding author
MN
ABSTRACT
OBJECTIVE: Diabetic nephropathy is the commonest cause of Chronic Kidney Disease in India. Use of urinary
microalbumin in screening is limited due to its high cost. The study aimed to determine optimal cut-off value of P:C
ratio for the prediction of microalbuminuria.
MATERIALS AND METHODS: 90 patients of type 2 diabetes with random urine samples showing ‘-‘ or ‘±’(traces) with dipstick test for proteins, were included. Urinary Microalbumin, and total urinary Protien to creatinine ratio were done on random urine samples.
RESULTS: 48 subjects had microalbuminuria and 42 subjects had normalbuminuria. Optimal cut-off value for PC Ratio for detection of
microalbuminuria was >0.08 with a sensitivity and specificity of 78.83 and 97.62 respectively.
CONCLUSION: Measurement of urinary total Protein to Creatinine Ratio is simple, inexpensive and showed strong positive correlation with
the Albumin Creatinine Ratio AND may be more efficient and cost effective than using the albumin to creatinine ratio.
INTRODUCTION
Diabetes mellitus type 2, a metabolic disorder characterized by
hyperglycemia due to insulin resistance and relative insulin deficiency, is associated with an increased risk of cardiovascular
disease and chronic kidney disease (CKD).1
Microalbuminuria can be defined as a urinary albumin excretion
of 30 to 300 mg/24 hours, if measured using a 24-hour urine collection, as urinary albumin excretion rate of 20 to 200 μg/min,
if measured in a timed urine collection, or 30 to 300 mg/g of
creatinine, if measured in a random, spot urine sample, called
as urinary albumin creatinine ratio (ACR) (in the absence of urinary tract infection and acute illness including myocardial infarction).2, 3 The ‘gold standard’ for the quantitative estimation of
urinary albumin and defining microalbuminuria is collection of
a 24-hour urine sample. However, 24-hour urine collections are
subject to error and poor compliance. The ACR is a more convenient test and is less prone to errors due to improper collection. 2, 3, 4
Microalbuminuria is an early sign of progressive renal and cardiovascular disease in general population as well as in patients
with diabetes mellitus and hypertension.5, 6 Once diabetic nephropathy develops, it eventually leads to renal insufficiency.3
Screening for microalbuminuria is recommended for all patients
with diabetes or kidney disease, because, at this stage, an attempt can be made to reverse diabetic nephropathy or slow its
progression, using suitable therapeutic interventions.3 The Kidney Disease: Improving Global Outcomes (KDIGO) CKD guidelines recommend that CKD should be classified based on the
cause, glomerular filtration rate and degree of albuminuria.7
However, the use of urinary microalbumin in screening for diabetic nephropathy is limited because of its high cost.
The amount of total protein in a spot, random urine sample may
be measured along with urine creatinine and reported as ratio
of urine protein to creatinine (PC Ratio).8 Urinary PC Ratio in a
spot urine sample has been used as an alternative to urine protein excretion in 24 hour urine samples.8 Can the urinary PC Ratio in spot urine samples be used to predict microalbuminuria?
The present study was undertaken to evaluate the diagnostic
value of the less expensive P:C ratio in spot voided urine samples for detection of the more expensive microalbuminuria in
patients with type 2 diabetes mellitus, and also to determine the
optimal cut-off value of P:C ratio with best sensitivity and specificity for the prediction of microalbuminuria.
MATERIALS AND METHODS
This study was conducted between Sepetmber 2012 and March
2013 at MS Ramaiah Medical College & Hospitals, Bangalore.
Ethics committee of the institute approved of the study and a
written informed consent was obtained from each participant
of the study. 90 subjects aged between 30 to 60 years, who were
patients with a diagnosis of type 2 diabetes of more than 1 year
duration, coming to the Department of Laboratory Medicine for
investigations namely Fasting Blood sugar, Serum Creatinine
and Glycated Hemoglobin (HbA1c), were included in the study.
Diagnosis of type 2 diabetes was made on the basis of history
and reports of laboratory investigations- Fasting Blood Sugar,
according to the WHO criteria {Fasting Serum Glucose ≥ 126
mg/dl, fasting was defined as no caloric intake for atleast 8-12
hours}. 1 Random spot urine samples of all the study subjects
were collected and only those samples showing ‘-‘ or ‘±’(traces),
with dipstick test for proteins were included in study. Patients
with dipstick test for proteins result ‘1+’ (30mg/dL or <0.5g/
day), ‘2+’ (100mg/dL or 0.5-1g/day), ‘3+’ (300mg/dL or 1-2g/day)
9, 10
or above, urinary tract infection, hypertension, chronic renal
failure, glomerular nephritis due to other systemic conditions,
febrile conditions, women menstruating at the time of sample
collection and pregnant women were excluded from the study. A
detailed history was taken along with blood pressure measurement and physical examination. 3ml of a fasting sample of blood
was collected into a plain vacutainer using due aseptic precautions, after confirming no calorific intake for atleast 8-10 hours.
The blood samples were allowed to clot and after centrifugation, the following biochemical investigations were done. Fasting
blood sugar(FBS) (by hexokinase method) using kits supplied by
Roche Diagnostics (Basel, Switzerland) on Cobas 6000 c501 RXL
MAX TM ,fully automated analyzer in the Biochemistry Laboratory of M S Ramaiah Hospitals. Glycated haemoglobin (HbA1c)
was done on a standardised Biorad D10 analyser using High Performance Liquid Chromatography. A random spot urine sample
was collected after withdrawal of blood sample and the following analysis was done on the urine samples- Albumin in urine
was estimated by Immunoturbidimetric assay 11, Urine protein
was estimated by turbidimetric method using benzethonium
chloride12, Urine creatinine by Buffered kinetic Jaffe’s reaction
without deproteinization13, on Cobas 6000 c501 RXL MAX TM in
the Biochemistry lab, Department of Laboratory Medicine at M
S Ramaiah Hospitals. The Protein Creatinine ratio was calculated and expressed as follows
Urinary Protein to Creatinine Ratio=
Urinary Protein in mg/dL ÷ Urinary Creatinine in mg/dL
IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH
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Research Paper
Volume : 3 | Issue : 9 | September 2014 • ISSN No 2277 - 8179
Urine Microalbumin was expressed in terms of mg/g of creatinine. This was calculated using the following formula:
Urinary Microalbumin = Urine albumin in mg/dL X 1000 ÷
Urine Creatinine in mg/dL
Creatinine Clearance was calculated for all the study subjects
based on the MDRD formula. 2
STATISTICAL ANALYSIS:
Quantitative variables such as age, urinary protein to creatinine,
urinary albumin were described in terms of Mean ± SD (MinMax) and results on categorical measurements are presented
in Number (%). Significance was assessed at 5 % level of significance. Student t test (two tailed, independent) was used to find
the significance of study parameters on continuous scale between two groups. Chi-square/ Fisher Exact test has been used
to find the significance of study parameters on categorical scale
between two or more groups. Pearson correlation was done to
find correlation between Urinary microalbuminuria with study
variables. A p value less than .05 was considered significant.
RESULTS:
90 subjects were included in the present study. The study subjects were divided into two groups based on the presence or absence of microalbuminuria- Group 1: Patients of Diabetes Mellitus having microalbuminuria (URINE ALBUMIN BETWEEN
30-300 mg/g of creatinine) comprising 48 subjects and Group 2:
Patients of Diabetes Mellitus having normalbuminuria (URINE
ALBUMIN BELOW 30mg/g of creatinine) comprising 42 subjects.
We did not have any subject with macroalbuminuria (URINE ALBUMIN ABOVE 300 mg/g) as we excluded subjects with results
for dipstick test for protein of ‘1+’ and above.14, 15 There were 31
(64.6%) males and 17 (35.4%) females in group 1 and 22(52.4%)
males and 20(47.6%) females in group 2. The mean age in years
±S.D was 51.46 ±8.79 in the microalbuminuria group (Group 1)
and 51.90±9.27 in the normoalbuminria group (Group 2). Baseline characteristics of patients are shown in table 1. There was
a statistically significant difference between the two groups in
terms of average duration of diabetes mellitus, calculated Creatinine Clearance, Fasting Blood Sugar (FBS), Post Prandial Blood
Sugar (PPBS), Glycated hemoglobin(HbA1c), Protein to creatinine ratio (PCR) and Urinary microalbumin.
Table 2 shows Pearson correlation of microalbuminuria with
variables like age, serum creatinine, creatinine clearance, FBS,
PPBS, HbA1c and PC Ratio. A significant linear relationship
was seen with urine microalbumin and calculated creatinine
clearance (negative correlation r=-0.770, p<0.001), FBS (r=0.397,
p<0.001), HbA1c (r=0.799, p<0.001) and protein to creatinine ratio (r=0.807, p<0.001) Refer figure1.
A Receiver Operating Characteristics curve analysis was done
for the urinary protein to creatinine ratio (PC Ratio) and urine
microalbumin. The optimal cut-off value for the PC Ratio for the
detection of microalbumin was >0.08 as shown in table 3 and
figure2.
DISCUSSION:
Chronic kidney disease (CKD) is a worldwide public health problem, due to the number of patients and cost of treatment involved. Early detection and treatment can prevent or delay complications like renal failure, cardiovascular disease or premature
death.2 Diabetic kidney disease is the commonest cause of CKD
in India.16 Diabetic nephropathy takes a downhill path from
normoalbuminuria (Albumin Creatinine Ratio ACR<30mg/g of
creatinine) to CKD which is marked by the middle stage microalbuminuria (ACR 30 – 300 mg/g of creatinine) progressing to
overt proteinuria ACR >300 mg/g of creatinine). 3, 14 There were
an estimated 40 million persons with diabetes in India in 2007
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and this number may increase to almost 70 million people by
2025 by which time every fifth diabetic subject in the world
would be an Indian.17 Adding to the economic burden is the high
prevalence of complications because of poorer control of diabetes and lack of access to diagnostics and health care, leading to
increased morbidity and mortality. 18 Urinary microalbuminuria,
as a screening tool for diabetic nephropathy is not being used
maximally all over India due to its high cost. A cross sectional
study was undertaken in order to determine if total protein to
creatinine ratio of a spot, random sample of urine which is a less
expensive method could be used to predict the presence of microalbuminuria in type 2 Diabetes mellitus patients.
The study subjects were divided into Group 1 having patients of
Diabetes Mellitus with microalbuminuria and Group 2 comprising patients of Diabetes Mellitus with normalbuminuria. The duration of diabetes, calculated creatinine clearance, Fasting Blood
Sugar, Glycated hemoglobin and urinary PC ratio were higher
in the microalbuminuric group when compared to the normalbuminuric group. This finding is similar to other studies. 19, 20,
21
A significant positive linear relationship was seen with urine
microalbumin and HbA1c (r=0.799, p<0.001) in group 1(microalbumiuria group) but not significant in group 2(normalbuminuria group). A positive correlation between microalbuminuria
and HbA1c has been found in other studies.20, 21 Glycosylation
of hemoglobin takes place in a spontaneous, non-enzymatic
manner, under physiological conditions by a reaction between
circulating glucose and N-terminal valine of beta chain of hemoglobin molecules to form glycated or glycosylated hemoglobin
(HbA1c). HbA1c levels indicate glycemic control and correlate
well with mean blood glucose levels over the previous eight to
twelve weeks.22 Higher levels of HbA1C have been associated
with increased risk for development of microangiopathy in diabetic. This could be because HbA1c has special affinity for oxygen thereby causing tissue anoxia and plays a role in causation
of micro and macroangiopathy. 23
Urinary microalbumin, measured in the present study as ACR
and urinary total PC Ratio also showed a significant positive
correlation (r=0.807, p<0.001). The Receiver-operating characteristics curve analysis showed that the protein to creatinine ratio
had sensitivity and a specificity of 78.83% and 97.62%, respectively, for the detection of microalbuminuria at a cuttoff value
of 0.08g/g of creatinine with an AUC (Area under the curve) of
0.881. A study done by Yamamoto K et al.24, which included 150
patients of type 2 diabetes mellitus, found that the protein to
creatinine ratio had a sensitivity and a specificity of 90.8% and
91.9 %, respectively, for the detection of microalbuminuria at a
cuttoff value of 0.09g/g of creatinine. In another study done by
Yamomoto K et al.25, that included 784 patients attending the
cardiovascular clinic, found a strong positive correlation between the Total PC Ratio and ACR (r= 0.861, p < 0.001) and PC
Ratio revealed a sensitivity of 94.4%, a specificity of 86.1%, for detecting microalbuminuria for a PC Ratio cut-off value of .084g/g
of creatinine, indicating that the cut-off value could be used for
patients with CVD risk factors.
An economical test for screening, the semiquantitative dipstick
analysis of urinary protein concentration, is used by general
practitioners. But, a physician is most likely to ignore a ‘-‘dipstick
result and a ‘±’(traces) dipstick result as the clinical significance
of ‘±’(traces) proteins on dipstick is not well defined. The present
study determined microalbuminuira in random spot urine samples showing ‘-‘ or ‘±’(traces), with dipstick test for proteins in
patients of type 2 diabetes mellitus. In our study, 42(46.66%) out
of the 90 diabetic study subjects who had ‘-‘ or ‘±’(traces) with
dipstick test for proteins, had microalbuminuria with ACR 30 –
300 mg/g of creatinine. This shows that urine dipstick test may
not have sufficient sensitivity and specificity to detect microalbuminuria. The measurement of urinary total Protein to Creati-
Research Paper
Volume : 3 | Issue : 9 | September 2014 • ISSN No 2277 - 8179
nine Ratio is simple, inexpensive and showed a strong positive
correlation with the Albumin Creatinine Ratio. Periodic screening of patients of type 2 diabetes mellitus for microalbuminuria
using the protein to creatinine ratio may be more efficient and
cost effective than using the albumin to creatinine ratio and may
enable early identification of progressive renal and cardiovascular diseases and therapeutic interventions leading to better outcome for the patient.
The major limitations of our study are the small sample size
and only patients with diabetes mellitus were included. Further
studies with a larger sample size need to be undertaken to determine whether similar results will be obtained in diabetics and
non diabetics with cardiovascular disease or even in the general
population.
Table 1: Baseline Characteristics of study subjects
Age in years
Group 1 Micro- albuminuria Group 2 Norm-albuminuria Total
Mean ± S.D
Mean ± S.D
Mean ± S.D
p value
51.46±8.79
51.90±9.27
51.67±8.97
0.815
Average duration of diabetes in months 70.6±5.6
47.9±6.2
59.25±5.8
0.01
Serum creatinine (mg/dL)
0.89±0.38
0.90±0.29
0.871
Creatinine clearance in mL/min/1.73m2 75.32±9.91
83.55±8.17
79.16±9.99
<0.001**
FBS (mg/dl)
177.83±78.18
136.52±23.91
158.56±62.61
0.001**
PPBS (mg/dl)
251.19±102.19
192.86±45.4
223.97±85.56
0.001**
HbA1c %
8.42±0.86
7.30±0.65
7.90±0.94
<0.001**
Protein to Creatinine ratio(PC Ratio)
0.15±0.11
0.05±0.03
0.10±0.09
<0.001**
10.32±7.45
80.44±89.6
<0.001**
0.90±0.17
Urinary microalbumin (mg/g creatinine) 141.78±83.16
Table 2: Pearson correlation of study variables with urine microalbumin
Pair
Micro albuminuria
Normalbuminuria
TOTAL
r value
p value
r value
p value
r value
p value
Urinary microalbumin mg/g vs S.Creatinine -0.003
mg/dL
0.984
-0.138
0.384
0.005
0.964
Urinary microalbumin mg/g vs Creatinine
clearance in mL/min/1.73m2
-0.905
<0.001**
-0.785
<0.001**
-0.770
<0.001**
Urinary microalbumin mg/g vs
FBS mg/dL
0.254
0.082+
-0.170
0.282
0.397
<0.001**
Urinary microalbumin mg/g vs
HbA1c%
0.804
<0.001**
0.213
0.176
0.799
<0.001**
Urinary microalbumin mg/g vs
PC Ratio
0.746
<0.001**
0.458
0.002**
0.807
<0.001**
Table 3: ROC curve analysis to determine the diagnostic value of Protein Creatinine Ratio to predict Microalbuminuria
PCR
Cut-off
Sensitivity
Specificity
LR+
LR-
AUC
P value
>0.08
78.83
97.62
29.75
0.30
0.881
0.001**
Figure 1: Correlation between Microalbumin (Albumin to
Creatinine ratio) and total urinary protein to creatinine ratio, serum creatinine, creatinine clearance and HbA1c in
Group 1(Microalbuminurics)
Figure 2: ROC curve analysis to determine the optimal cutoff value of urinary Protein to Creatinine Ratio to predict
Microalbuminuria
PCR
100
Scatterplot in Micro albuminuria
PCR
Serum_creatinine
80
400
200
100
0
0.00
0.15
0.30
0.45
Creatinine_clearance
0.60
0.50
0.75
1.00
HbA1c
1.25
1.50
60
Sensitivity
urinary microalbuminuria
300
40
400
300
20
200
100
0
0
0
60
70
80
90
100 7
8
9
10
11
20
40
60
80
100
100-Specificity
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Volume : 3 | Issue : 9 | September 2014 • ISSN No 2277 - 8179
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