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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 46  |  Issue : 2  |  Page : 145-151

Prospective randomized study to evaluate urinary liver-type fatty acid binding protein in early detection of diabetic nephropathy in type 2 diabetic patients


1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission01-Jun-2017
Date of Acceptance18-Dec-2017
Date of Web Publication31-Oct-2018

Correspondence Address:
Eeem Awny
Clinical Pathology Department, Faculty of Medicine, Tanta University, El-Gharbia, El Mahalla El Koubra, Manshyet El Bakry, 8 Suez Street, 31159
Egypt
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DOI: 10.4103/tmj.tmj_54_17

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  Abstract 


Background The early diagnosis of diabetic nephropathy (DN) is important to avoid the adverse outcomes of renal failure among diabetics. Although microalbuminuria has been recognized as a significant predictive marker for the early diagnosis of DN, some diabetics may develop DN without an apparent microalbuminuria. Therefore, sensitive and specific markers are required to detect DN in its early stages.
Aim The aim of this work was to estimate the liver-type fatty acid binding protein (L-FABP) level in type 2 diabetes mellitus (T2DM) patients with DN and shed more light on its value and clinical significance.
Materials and methods The present study was carried at the Clinical Pathology Department in Tanta University. It included 30 patients with T2DM, who attended the Outpatient Clinic of Internal Medicine Department, Tanta University Hospital, and fulfilled the criteria of the American Diabetes Association, 2015. The control group included 20 healthy individuals. The participants were subdivided into the following groups: group 1 included 10 T2DM patients with normoalbuminuria, eight women and two men. Their ages ranged from 55 to 75 years. Group 2 included 10 T2DM patients with microalbuminuria, five women and five men. Their ages ranged from 45 to 72 years. Group 3 included 10 T2DM patients with macroalbuminuria, four women and six men. Their ages ranged from 49 to 67 years. Group 4 included healthy individuals who served as a control group, nine women and 11 men. Their ages ranged from 43 to 75 years.
Results The present study found that there was no significant difference between the three patient groups in disease duration, fasting, and postprandial blood glucose. There was a significant difference between the three patient groups in glycated hemoglobin, albumin in urine, glomerular filtration rate, and serum creatinine. There was a significant difference in L-FABP between the three patient groups and the control group.
Conclusion Urinary L-FABP level was significantly increased in diabetic patients with DN compared with the control group. The levels of urinary L-FABP in each DN group were significantly increased according to the severity of DN. The high levels of L-FABP in urinary excretion were associated with deteriorating renal function in patients with T2DM. This association was frequently observed even in patients with normoalbuminuria

Keywords: diabetes, diabetic nephropathy, liver-type fatty acid binding protein, microalbuminuria


How to cite this article:
Awny E, Elwan N, Okasha K, Suliman GA. Prospective randomized study to evaluate urinary liver-type fatty acid binding protein in early detection of diabetic nephropathy in type 2 diabetic patients. Tanta Med J 2018;46:145-51

How to cite this URL:
Awny E, Elwan N, Okasha K, Suliman GA. Prospective randomized study to evaluate urinary liver-type fatty acid binding protein in early detection of diabetic nephropathy in type 2 diabetic patients. Tanta Med J [serial online] 2018 [cited 2018 Nov 21];46:145-51. Available from: http://www.tdj.eg.net/text.asp?2018/46/2/145/244687




  Introduction Top


Diabetes mellitus (DM) is a heterogeneous disturbance of the metabolism in which the main finding is chronic hyperglycemia. The cause is either impaired insulin secretion or impaired insulin action, or both. Chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels [1],[2].

Type 2 diabetes mellitus (T2DM) is the predominant form of diabetes and previously referred to as noninsulin-dependent diabetes or adult-onset diabetes that occurs in individuals who have insulin resistance and usually have relative rather than absolute insulin deficiency. The risk of developing this form of diabetes increases with age, obesity, and lack of physical activity. It occurs more frequently in women with previous gestational gestational diabetes mellitus (GDM) and individuals with hypertension or dyslipidemia, and its frequency varies in different racial/ethnic subgroups. It is often associated with a strong genetic predisposition, rather than the autoimmune form of type 1 diabetes. In T2DM, usually, there are no complains from the patient [3].

Diabetic nephropathy (DN), a renovascular complication that affects nearly 30% of diabetics and leads to an end-stage renal disease, is a common microvascular complication of T2DM. It known as Kimmelstiel–Wilson syndrome or nodular diabetic glomerulosclerosis or intercapillary glomerulonephritis. It is a clinical syndrome characterized by albuminuria [>300 mg/g creatinine (Cr)], confirmed on at least two occasions 3–6 months apart, a permanent and irreversible decrease in the glomerular filtration rate (GFR), and arterial hypertension [4],[5].

The early diagnosis of DN is important to avoid the adverse outcomes of renal failure among diabetics. Although microalbuminuria has been recognized as a significant predictive marker for the early diagnosis of DN, some diabetics may develop DN without an apparent microalbuminuria. Therefore, sensitive and specific markers are required to detect DN in its early stages [6],[7].

Liver-type fatty acid binding protein (L-FABP), also known as FABP1, an intracellular carrier protein of free fatty acids, is expressed in the liver and the kidney. In the kidney, the expression of L-FABP is predominantly located in the proximal tubules. The high levels of urinary L-FABP were previously suggested to be associated with renal tubulointerstitial damage because excessive reabsorption of free fatty acids into the proximal tubules induces tubulointerstitial damage [8]. However, whether L-FABP would be a more sensitive marker of DN than albumin excretion rate (AER) or whether its predictive role is solely confined to the progression of the disease process is not yet known. Therefore, it is important to investigate whether baseline levels of L-FABP predict the development of DN and its progression at any stage of the disease and whether the use of L-FABP alone or together with AER is beneficial compared with current standard testing by AER [9].


  Aim Top


The aim of this work was to estimate the L-FABP level in T2DM patients with DN and shed more light on its value and clinical significance.


  Materials and methods Top


After obtaining approval from the research ethics committee and informed written consent from the patients, the present study was carried at the Clinical Pathology Department in Tanta University. It included 30 patients with T2DM, who attended the Outpatient Clinic of the Internal Medicine Department, Tanta University Hospital, and fulfilled the criteria of American Diabetes Association, 2015. The control group included 20 healthy individuals.

The participants were subdivided as follows:
  • Group 1: included 10 T2DM patients with normoalbuminuria, eight women and two men. Their ages ranged from 55 to 75 years.
  • Group 2: included 10 T2DM patients with microalbuminuria, five women and five men. Their ages ranged from 45 to 72 years.
  • Group 3: included 10 T2DM patients with macroalbuminuria, four women and six men. Their ages ranged from 49 to 67 years.
  • Group 4: healthy cases served as a control group, nine women and 11 men. Their ages ranged from 43 to 75 years.


All patients were subjected to the following:
  1. Detailed assessment of history, with a special focus on age, sex, symptoms of diabetes mellitus, as well as duration of the disease.
  2. Clinical examination: All patients were examined to exclude systemic and local diseases other than diabetes mellitus or renal disease.
  3. Laboratory investigations included the following:
    • Serum level of fasting and postprandial blood glucose.
    • Serum level of Cr.
    • Glycated hemoglobin A1c (HbA1c).
    • Albumin in urine.
  4. Specific laboratory investigations:-
    • Estimation of urinary L-FABP by the enzyme-linked immune-sorbent assay (ELISA) technique.


Methods

Sample collection
  1. Spot urine samples were collected in sterile containers, centrifuged for 20 min at a speed of 2000–3000 rpm, and then the supernatant was removed and kept at −20°C for use.
  2. Eight milliliter of peripheral venous blood was obtained from every participant using a completely aseptic technique. The samples were divided as follows: 2 ml in an EDTA (1.5–2 mg/ml blood) tube for HbA1c and 6 ml in plain tubes for centrifugation. The serum was separated for other investigations.


Serum glucose assay

Glucose was determined by enzymatic oxidation in the presence of glucose oxidase. The hydrogen peroxide formed reacts under the effect of peroxidase with phenol and 4-aminoantipyrine to form a red violet quinoneimine as an indicator. The intensity of color formed was proportional to the glucose concentration in the sample [10]. The level of glucose was determined by measuring the absorbance at 546 nm using an ERMA Inc. (Yushima, Bunokyo, Tokyo, Japan) photometer model AE-600n; comparison with the standard was performed.

Serum creatinine assay

The assay is based on the reaction of Cr with sodium picrate (Jaffe reaction). Cr reacts with alkaline picrate, forming a red complex. The intensity of color formed is proportional to the Cr concentration in the sample [11]. The level of serum Cr was determined by measuring the absorbance at 492 nm using an ERMA Inc. photometer model AE-600n, the ratio of absorbance was calculated, and comparison with the standard was performed.

Glycated hemoglobin

Hemolyzed whole blood was mixed with a weakly binding cation exchange resin. The non-HbA1c bonded to the resin, whereas the HbA1c remained and was removed using a resin separator in the supernatant. The percent of HbA1c was determined by measuring the absorbance at 405 nm of the HbA1 fraction and the total Hb fraction using high-performance liquid chromatography model Tosoh G8-90sl (Tosoh Corporation, Shipa, Minato, Tokyo, Japan), the ratio of absorbance was calculated, and comparison with the standard was performed [12].

Urinary albumin/creatinine ratio

Urinary albumin was measured by a quantitative assay using antibodies to human albumin measured using the immunoturbidimetric method at wavelengths of 340 and 700 nm and then comparison of absorbance with the standard was performed [13]. Urinary Cr concentration was measured using the colorimetric method at a wavelength of 492 nm. The ratio of absorbance was calculated, comparison with the standard was performed, and the albumin Cr ratio was calculated [14]. Both urinary albumin and urinary Cr absorbance were measured using an ERMA Inc. photometer model AE-600n.

Estimated glomerular filtration rate

A formula called the Cockcroft–Gault equation has been developed to predict Cr clearance from serum Cr. The Cockcroft–Gault equation is as follows [15]:



Method of liver-type fatty acid binding protein assay

L-FABP was estimated in urine using the ELISA kit by SunRed Company (Shanghai, China).

Principle of the assay

The kit used a double-antibody sandwich ELISA to measure the level of human L-FABP in urine. L-FABP was added to a monoclonal antibody enzyme well that was precoated with human L-FABP monoclonal antibody and incubated; then, (L-FABP) antibodies labeled with biotin were added and combined with streptavidin-HRP to form an immune complex. Incubation was performed, followed by washing to remove the uncombined enzyme. Then, chromogen solutions A, B were added, the color of the liquid changed into blue, and because of the effect of acid, the color finally became yellow. The chrima of the colour and the concentration of L-FABP of the sample were positively correlated.

Assay procedure

Standard dilution

This test kit supplied one original standard reagent that was diluted into five standards of different concentrations by serial dilution as follows:



Inject samples

  1. Blank well: no sample or standard was added.
  2. Standard wells: 50 µl standard was added; followed by 50 µl of streptavidin-HRP (as the standard already included biotin antibody, it was not necessary to add the antibody).
  3. To test wells: 40 µl from each sample was added, and then we added both L-FABP-antibody 10 µl and streptavidin-HRP 50 µl. The sealing membrane was sealed and shaken gently. Then, it was incubated for 60 min at 37°C.
  4. Washing solution was prepared: It was diluted 30 times with distilled water as a standby.
  5. The membrane was removed carefully and the liquid was drained. The remaining water was discharged.
  6. Chromogen solution A, 50 µl, was added and then 50 µl of chromogen solution B was added to each well. It was mixed gently and incubated for 10 min at 37°C away from light.
  7. Stop: 50 µl of stop solution was added to each well to stop the reaction (the blue changed into yellow immediately).
  8. Final measurement: the blank well was taken as zero and the optical density (OD) was measured under 450 nm wavelength within 15 min after the stop solution was added.
  9. According to standards’ concentration and the corresponding OD values, the standard curve was calculated and then the OD values of the samples were used to calculate the corresponding sample’s concentration.



  Results Top


[Table 1] shows the fasting blood glucose in the three patient groups; in group I, the range was 150–260 mg/dl, with a mean value of 198.5±32.66 mg/dl, in group II, the range was 190–260 mg/dl, with a mean value of 222.5±20.45 mg/dl, and in group III, the range was 204–330 mg/dl, with a mean value of 245.8±43.91 mg/dl. There was no statistically significant difference in fasting blood glucose between the three patient groups.
Table 1 Comparison between the three patient groups in fasting blood glucose

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[Table 2] shows HbA1c in the three patient groups; in group I, the range was 6.9–8.1%, with a mean value of 7.58±0.40%, in group II, the range was 8.3–9.6%, with a mean value of 8.98±0.51%, and in group III, the range was 11.3–13.0%, with a mean value of 12.32±0.076%. There was a statistically significant difference in HbA1c between the three patient groups (P<0.05).
Table 2 Comparison between the three patient groups in glycated hemoglobin

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[Table 3] shows urinary albumin (mg/g Cr) in the three patient groups; in group I, the range was 10–28 mg/g Cr, with a mean value of 20.62±4.99 mg/g Cr, in group II, the range was 105–290 mg/g Cr, with a mean value of 163.6±65.27 mg/g Cr, and in group III, the range was 250–610 mg/g Cr, with a mean value 463.0±144.6 mg/g Cr. There was a statistically significant difference in urinary albumin between the three patient groups (P<0.05).
Table 3 Comparison between the three patient groups in urinary albumin

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[Table 4] shows the estimated glomerular filtration rate (eGFR) (ml/min/1.73 m2) in the three patient groups; in group I, the range was 79–102 ml/min/1.73 m2, with a mean value of 92.1±8.10 ml/min/1.73 m2, in group II, the range was 32–55 ml/min/1.73 m2, with a mean value of 43.1±7.68 ml/min/1.73 m2, and in group III, the range was 21–29 ml/min/1.73 m2, with a mean value 24.60±3.57 ml/min/1.73 m2. There was a statistically significant difference in the estimated glomerular filtration rate between the three patient groups (P<0.05).
Table 4 Comparison between the three patient groups in the estimated glomerular filtration rate

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[Table 5] shows serum Cr (mg/dl) in the three patient groups; in group I, the range was 0.9–1.3 mg/dl, with a mean value of 1.08 ±0.13 mg/dl, in group II, the range was 1.4–1.9 mg/dl, with a mean value of 1.62±0.18 mg/dl, and in group III, the range was 2–3.10 mg/dl, with a mean value 2.44±0.39 mg/dl. There was a statistically significant difference in serum Cr between the three patient groups (P<0.05).
Table 5 Comparison between the three patient groups in serum creatinine

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[Table 6] shows L-FABP in the three patient groups and the control group; in group I, the range was 298–398 ng/ml, with a mean value of 356.1±26.04 ng/ml, in group II, the range was 407–537 ng/ml, with a mean value of 440.5±38.43 ng/ml, and in group III, the range was 524–731 ng/ml, with a mean value 594.4±75.98 ng/ml, whereas the range in the control group was 181–315 ng/ml, with a mean value of 230.0±35.49 ng/ml. There was a statistically significant difference in L-FABP between the three patient groups and the control group (P<0.05).
Table 6 Comparison between the three patient groups and the control group in liver-type fatty acid binding protein

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There was a significant positive correlation between L-FABP and HbA1c, urinary albumin, and serum Cr in diabetic patients as shown in [Figure 1],[Figure 2],[Figure 3]. However, there was a significant negative correlation between L-FABP and eGFR as shown in [Figure 4].
Figure 1 Scatter diagram showing a positive correlation between liver-type fatty acid binding protein (L-FABP) and glycated hemoglobin (HbA1c) in patient groups.

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Figure 2 Scatter diagram showing a positive correlation between liver-type fatty acid binding protein (L-FABP) and urinary albumin in patient groups.

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Figure 3 Scatter diagram showing a positive correlation between liver-type fatty acid binding protein (L-FABP) and serum creatinine (S. Cr) in patient groups.

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Figure 4 Scatter diagram showing a negative correlation between liver-type fatty acid binding protein (L-FABP) and estimated glomerular filtration rate (eGFR) in patient groups.

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  Discussion Top


L-FABP is expressed in the liver and kidney. In the kidney, the expression of L-FABP is predominantly located in the proximal tubules. Fatty acids induce the expression of L-FABP in the proximal tubules; thus, urinary L-FABP in humans may be a useful clinical marker that can help predict and monitor the progression of renal dysfunction in patients with T2DM [8],[16].

This study investigated whether baseline levels of L-FABP predict the development of DN and its progression at any stage of the disease and whether the use of L-FABP alone or together with AER is beneficial compared with current standard testing by AER.

In terms of L-FABP, there was a highly statistically significant difference in the L-FABP levels between normoalbuminuria patients, microalbuminuria patients, and macroalbuminuria patients. Also, there was a highly statistically significant difference between the three patient groups and the healthy control group in L-FABP. This was in agreement with Kamijo Ikemori et al. [17], who found that urinary levels of L-FABP and urinary albumin in the patients with normoalbuminuria were significantly higher than those in normal control participants. Also, the levels of urinary L-FABP and urinary albumin in each DN group were significantly different from the levels in all of the other groups and increased significantly according to the severity of DN.

Shin-Ichi et al. [16] also reported that urinary levels of L-FABP in patients with microalbuminuria were higher than in those with normoalbuminuria and the high levels of L-FABP in urinary excretion were associated with deteriorating renal function in patients with T2DM. This association was frequently observed even in patients with normoalbuminuria.

In the present study, there was a significant positive correlation between serum L-FABP and serum Cr, HbA1c, and urinary albumin, whereas there was a negative correlation between serum L-FABP and eGFR.

This was in agreement with the results of Kumi et al. [18] as they reported that urinary albumin was proportionally correlated with urinary L-FABP and GFR was inversely correlated with urinary L-FABP. Our results were also in agreement with those of Naohi et al. [19] as they reported that urinary L-FABP levels correlated positively with urinary albumin and negatively with eGFR.

In addition, Gudeta, et al. [20] reported that there was a strong positive correlation between albumin/Cr and the L-FABP level. Also, a positive correlation was reported between Urinary L-FABP level and serum Cr by Kamijo et al. [21]. The previous reports are in agreement with our results, showing that urinary L-FABP levels correlated with the progression of renal impairment. It could be a reliable and more sensitive marker for renal damage in DN.


  Conclusion Top


Urinary L-FABP level was significantly increased in diabetic patients with DN compared with the control group. The levels of urinary L-FABP in each DN group were significantly increased according to the severity of DN. The high levels of L-FABP in urinary excretion were associated with deteriorating renal function in patients with T2DM. This association was frequently observed even in patients with normoalbuminuria.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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