Intestinal protein loss in patients with haemorrhagic fever with renal syndrome

Young Ok Kim, Chul Woo Yang, Sun Ae Yoon, Ho Cheol Song, Yong Soo Kim, Suk Young Kim, Euy Jin Choi, Yoon Sik Chang and Byung Kee Bang

Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea



   Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. In haemorrhagic fever with renal syndrome (HFRS) vascular dysfunction has been observed in various organs, but the involvement of the intestine has not yet been reported. This study was performed to evaluate the association of intestinal protein loss in this disease with other clinical parameters reflecting vascular permeability or disease severity.

Methods. Twenty patients with HFRS were included in this study. Intestinal protein loss was measured by 99mTc-human serum albumin (99mTc-HSA) scintigraphy in the acute stage, and quantitative analysis of protein loss was measured by the faecal clearance of alpha 1-antitrypsin (CAT) in the acute and the recovery stages. CAT was then compared with clinical parameters reflecting disease activity and vascular permeability.

Results. 99mTc-HSA scintigraphy was positive in 13 (65%) patients, and CAT in the acute stage was significantly increased as compared with CAT in the recovery stage (40.5±24.1 vs 9.2±4.2 ml/day, P<0.001). CAT was associated with serum albumin levels, frequency of hypotensive episodes, severity of acute renal failure, and degree of thrombocytopenia.

Conclusions. Our data suggest that the increased vascular permeability of HFRS is associated with the increased intestinal loss of plasma proteins, which might represent one of the parameters of disease severity in HFRS.

Keywords: haemorrhagic fever; renal syndrome; hypoalbuminaemia; intestinal tract



   Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Haemorrhagic fever with renal syndrome (HFRS) is an acute inflammatory disease caused by Hantavirus [14] and characterized by vascular dysfunction [58]. Clinically, retroperitoneal oedema and free fluid accumulations in the body cavities with haemoconcentration reflect the vascular leak present in HFRS. Evidence of the vascular leak is frequently observed in patients dying of hypotensive shock [7,8]. This vascular leak may be associated with low serum protein values [7,8].

Protein loss via the intestinal tract has been well documented in diseases associated with vasculitis, such as systemic lupus erythaematosus and mixed connective tissue disease, and is explained by increased vascular permeability due to intestinal vasculitis [9,10]. Since increased vascular permeability is present in HFRS, increased protein loss via the intestinal tract is possible.

We evaluated the intestinal protein loss in HFRS. First, we confirmed intestinal protein loss by abdominal scintigraphy using technetium-99m-labelled human serum albumin (99mTc-HSA) and faecal clearance of alpha1-antitrypsin (CAT) [1114]. Secondly, we compared the intestinal protein loss with clinical parameters representing the disease severity.



   Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient population
Twenty patients with HFRS were included in this study. The diagnosis of HFRS was based on clinical manifestations (high fever, back pain, acute renal failure, proteinuria or haematuria, and thrombocytopenia) and indirect immunofluorescence antibody test against the Hantavirus. Fifteen patients were male and five were female. The mean age was 35±8 years (range 20–52 years). None of the subjects had a medical history of protein-losing enteropathy, such as inflammatory bowel disease.

Measurement of clinical and laboratory parameters
The association of the intestinal protein loss with clinical parameters (hypotension, pleural effusion, haemodialysis treatment) was also investigated. Blood pressure was measured with manometer; hypotension was defined as <90/60 mmHg. Pleural effusion was diagnosed by chest X-ray.

On admission, blood samples were obtained for complete blood count, creatinine, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), and a 24-h urine was collected for protein measurement. Acute renal failure was defined as serum creatinine >2.0 mg/dl. Leukocytosis was defined as leukocyte count >10 000/mm3, thrombocytopenia as platelet count <100 000/mm3, and hypoalbuminaemia as serum albumin level <3.5 g/dl.

Haemodialysis was performed in cases of severe pulmonary oedema, in oliguric or anuric states, and in cases of severe azotaemia (serum creatinine >10 mg/dl or blood urea nitrogen >100 mg/dl).

Abdominal scintigraphy using 99mTc-HSA
99mTc-HSA was performed within 7 days after the onset of symptoms. Twenty millicuries of 99mTc-HSA (Daiichi Radioisotope Corp, Japan) were intravenously injected, and scintigraphic images were obtained at 10 and 30 min, and at 2, 4, and 24 h after injection. Images of the thyroid were obtained at the same time that free technetium was measured. Protein loss via the intestinal tract was confirmed by the movement of the extravasated radioactive substance down the intestinal tract on abdominal scintigraphy within 24 h.

Fecal clearance of CAT
CAT was measured by nephelometer (Behring, Germany). Patients who could not defecate daily received oral magnesium hydroxide (1.5 g/day) to induce defecation. Faeces were collected for 24 h and kept refrigerated at 4°C. Two grams of faeces mixed with 2 ml of distilled water were centrifuged at 3500 r.p.m. for 10–20 min; the supernatant was used for the measurement of CAT. Serum concentration of AT was simultaneously measured by the same method and the clearance was calculated using the following formula:

Normal range of CAT was defined as 0–13 ml/24 h [11,12]. CAT was calculated in both the acute and the recovery stages in which serum albumin level had been normalized (>=3.5 g/dl) and renal function stabilized.

Statistical analysis
For comparison of the CAT in acute and recovery stages, paired Student's t-test was used; for comparison of the clinical factors between the groups of increased and normal CAT, unpaired Student's t-test and chi-square test were applied. The correlation between the serum albumin level, CAT, and the amount of proteinuria was evaluated by Pearson's correlation test; statistical significance was defined as P<0.05.



   Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Clinical and laboratory findings
Indirect immunofluorescence antibody tests were positive in all patients. IgG against Hantavirus was 3+ (1 : 512) in 13 patients, 2+ (1 : 128) in six patients, and 1+ (1:32) in one patient. Acute renal failure developed in all patients, and 12 (60%) patients needed haemodialysis. Both hypotension and pleural effusion developed in seven (35%) patients. Ascites and mild diarrhoea were observed in four (20%) and one (5%) patients, respectively. Hypoalbuminaemia was detected in 17 (85%) patients; serum albumin level during the acute stage was 3.0±0.3 g/dl. Leukocytosis and thrombocytopenia were observed in 15 (75%) and 19 (95%) patients, respectively. Proteinuria was found in all patients on admission (1.9±1.3 g/day), but was normalized within 7 days. Other laboratory data are listed in Table 1Go.


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Table 1. Laboratory findings in 20 patients with HFRS

 

Results of 99mTc-HSA scintigraphy
No radionuclide uptake by the thyroid was observed within 24 h. Thirteen patients (65%) had evidence of radioactive tracer moving down the intestinal tract, demonstrating protein loss into the intestinal lumen. Radioactivity was frequently observed in the right colon, but infrequently in the transverse and the left colon. Figure 1Go shows scintigraphic findings 24 h after the intravenous injection of 99mTc-HSA, with positive (Figure 1aGo) and negative (Figure 1bGo) results.



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Fig. 1. Abdominal scintigraphy 24 h after intravenous injection of 99mTc-HSA. Representative findings in patients with increased CAT (a) and in those with normal CAT (b). The patient with increased CAT showed radioactivity in the large intestinal tract including right, transverse, and left colons. In contrast, such intestinal radioactivity was not apparent in the patient with normal CAT.

 

Results of CAT
Increased CAT in the acute stage (40.5±24.1 ml/day) was observed in 13 (65%) patients, and significantly decreased CAT was noted in the recovery stage, as compared with CAT in the acute stage (9.2±4.2 ml/day, P<0.001) (Figure 2Go).



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Fig. 2. Comparison of CAT among patients with increased CAT during the acute and recovery stages. CAT in the recovery stage was measured when serum albumin level was normalized (>=3.5 g/dl). CAT in the recovery stage was significantly decreased, as compared with that in the acute stage (9.2±4.2 vs 40.5±24.1 ml/day, P<0.001).

 

Relationship between 99mTc-HSA scintigraphy and CAT
Of the 13 patients with increased CAT, 99mTc-HSA scan was positive in 12 and negative in one (Table 2Go).


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Table 2. The results of CAT and HSA scintigraphy

 

Comparison of clinical factors between normal and increased CAT groups
Hypotension developed in seven patients (53.8%) with increased CAT, but there were no episodes of hypotension in patients with normal CAT (P=0.016). The incidence of pleural effusion in the increased CAT group tended to be higher than in the normal CAT group, but there was no statistical difference between these two groups (46.1 vs 14.2%, P=0.154). Haemodialysis treatment was needed in 10 patients (76.9%) from the increased CAT group and in only two patients (28.5%) patients from the normal CAT group (P=0.035). The lowest level of serum albumin in the group with increased CAT was significantly lower than that in the group with normal CAT (2.8±0.1 vs 3.4±0.2 g/dl, P=0.001). The platelet count was lower in the patients with increased CAT, as compared to that in the patients with normal CAT (27 000±22 700 vs 76 000±80 000/mm3, P=0.049). Haemoglobin (17.3±2.4 vs 14.9±1.5 g/dl, P=0.032) and serum ALT (119±96 vs 53±23 IU/L, P=0.032) were significantly increased in the increased CAT group, as compared with those in the normal CAT group (Table 3Go).


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Table 3. Comparison of clinical parameters between normal and increased CAT groups

 

Correlation of the serum albumin level with CAT
There was a significant negative correlation between the lowest level of serum albumin during admission and CAT (r=-0.554, P=0.011), but there was no correlation between the lowest level of serum albumin and the amount of proteinuria on admission (r=0.062, p=0.638).



   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our study clearly demonstrates that protein loss via the intestinal tract occurs in the acute stage of HFRS. The intestinal protein loss, demonstrated by 99mTc-HSA scintigraphy and CAT levels showed a significant increase in the acute stage, and follow-up CAT in the recovery stage returned to the normal range. To our knowledge, this is the first report demonstrating that protein loss via the intestinal tract is increased in HFRS.

To evaluate the association of intestinal protein loss with increased vascular permeability in HFRS, we compared the CAT with other clinical parameters (hypotension, pleural effusion, haemoconcentration, thrombocytopenia). It is well known that hypotension, pleural effusion, and haemoconcentration are the parameters of increased vascular permeability in HFRS [7,8]. Potential mechanisms of thrombocytopenia include immune pathways leading to vascular endothelial injury and intravascular coagulation. Therefore, the degree of thrombocytopenia has been suggested as a clinical parameter of increased vascular permeability due to vascular dysfunction [7]. In this study, higher incidences of hypotension, pleural effusion, and haemoconcentration were associated with increased CAT. The degree of thrombocytopenia was also associated with increased CAT. These findings all suggest that intestinal protein loss is one of the results of increased vascular permeability in HFRS.

Our study was extended to determine whether intestinal protein loss represented the disease severity of HFRS. Serum albumin level, LDH, and the degree of renal failure have all been suggested as parameters of the disease severity in HFRS [7,8]. Haemodialysis treatment was needed in 80% of the patients with increased CAT, while only two (28.5%) with normal CAT underwent haemodialysis. Patients with increased CAT showed significantly low serum albumin levels, as compared with those with normal CAT. In addition, there was a good correlation between CAT and serum albumin level. The LDH levels also tended to be higher in the increased CAT group than in the normal CAT group. These findings together suggest that intestinal protein loss is closely associated with the disease severity of HFRS.

In general, hypoalbuminaemia can be attributed to the conditions of decreased synthesis (deficient protein intake, liver disease), excessive protein loss, or increased catabolism [15]. In HFRS, hypoalbuminaemia is frequently observed in the acute stage of HFRS [4], and its mechanism seems to be multifactorial, e.g., increased catabolism of protein, proteinuria, poor oral intake, and hepatic dysfunction. Therefore, it is difficult to define the exact cause of hypoalbuminaemia. However, the high correlation between intestinal protein loss and serum albumin level suggests that the intestinal protein loss partly contributed to hypoalbuminaemia. Further studies are needed to define other factors leading to hypoalbuminaemia.

In general, increased intestinal protein loss could be associated with increased water loss (such as diarrhoea) via the intestinal tracts In the acute stage of HFRS, abdominal pain, nausea, and vomiting are frequently observed, but diarrhoea is rarely observed [2,4,16]. In this study, diarrhoea was observed in only one patient (5%). Furthermore, some patients needed magnesium hydroxide to induce defecation. Therefore, it is less likely that episodes of diarrhoea have any significant impact on intestinal protein loss.

In this study, we demonstrated the intestinal protein loss in HFRS, but its actual pathophysiologic mechanism remains unknown. Potential mediators, which increase vascular permeability during the acute stage of HFRS, are tumor necrosis factor (TNF)-alpha, interleukin 1 and 2, and nitric oxide [5,7]. Green et al. [17] reported that the level of TNF-alpha was related to the degree of pleural effusion, representing the increased vascular permeability in patients with dengue haemorrhagic fever. Therefore, it can be postulated that the vascular injury and the subsequent increased production of the aforementioned cytokines may be responsible for the intestinal protein loss in HFRS.

99mTc-HSA scintigraphy has been utilized as a screening test for the intestinal protein loss because of its non-invasive and simple technique [13,14,18]. In addition, 99mTc-HSA is highly stable in vivo, and thus the radioactivity found in the intestines directly signifies the intestinal loss of 99mTc-HSA. The AT (molecular weight: 50 000 Da) is also useful in detecting intestinal protein loss [12,19,20]. The results of the 99mTc-HSA scintigraphy and the CAT levels were in concordance in our study: 99mTc-HSA scan and CAT were both positive in 12 out of the 20 patients, both negative in six patients. These two studies were in discordance in only two patients (Table 2Go). To exclude false-positive results, patients with gastrointestinal haemorrhage and inflammatory bowel disease were excluded from this study. Non-uptake of free technetium in the thyroid gland was used as a guide for excluding misinterpretation of the scintigraphic imaging results.

In conclusion, our study demonstrates that intestinal loss of plasma proteins is frequently observed in patients with HFRS and that it is closely associated with the clinical parameters reflecting increased vascular permeability and disease severity of HFRS.



   Notes
 
Correspondence and offprint requests to: Dr Byung Kee Bang, Department of Internal Medicine, Kangnam St Mary's Hospital, Catholic University Medical College, 505 Banpo Dong, Seocho Ku, Seoul 137–701, Korea. Back

* Preliminary results were presented at the 36th European Renal Association-European Dialysis and Transplant Association congress (Madrid, Spain) in 1999. Back



   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 1.12.99
Revision received 12. 4.00.



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