Serum leucine aminopeptidase as an activity indicator in systemic lupus erythematosus: a study of 46 consecutive cases

S. Inokuma, K. Setoguchi, T. Ohta, Y. Matsuzaki and A. Yoshida

Department of Allergy and Immunological Diseases, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan

Correspondence to: S. Inokuma, Department of Allergy and Immunological Diseases, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To determine whether elevations in serum leucine aminopeptidase (LAP) levels reflected the underlying evolution of active disease in systemic lupus erythematosus (SLE).

Methods. We studied serum LAP levels, other laboratory indicators, and SLE Disease Activity Index (SLEDAI) scores, in 46 consecutive patients with SLE admitted to Tokyo Metropolitan Komagome Hospital. LAP levels in 46 patients with rheumatoid arthritis were also measured.

Results. Thirty-three SLE patients had elevated LAP levels. LAP levels correlated positively with levels of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and {gamma}-glutamyl transpeptidase, and negatively with the total serum haemolytic complement and leucocyte, neutrophil and lymphocyte counts, but showed no correlation with alkaline phosphatase, {gamma}-globulin, ß2-microglobulin or C-reactive protein levels, or platelet count. The SLEDAI score correlated positively with LAP levels. The LAP level in patients with rheumatoid arthritis was near normal.

Conclusion. The serum LAP level may be a potential activity indicator for SLE.

KEY WORDS: LAP, SLE, Disease activity, SLEDAI, Lymphocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) exhibits a variety of clinical features, including exacerbations and remissions. It can be difficult to determine whether the disease is active or stable, and differentiating an exacerbation of the primary disease from other complications is also complicated. We have recently treated patients with active SLE who showed extremely high serum levels of leucine aminopeptidase (LAP), as measured by using leucinamide as the substrate.

Until recently, serum LAP levels have been thought to increase only as a manifestation of a liver/biliary tract disorder. This is because most of the substrates used thus far to test for LAP have been synthetics, such as L-leucyl-ß-naphthyl amide or L-leucyl-p-nitroanilide. When using these synthetic substrates, arylamidase (EC 3.4.11.2, `so-called LAP'), also called microsomal or particle-bound aminopeptidase, is mainly measured with very high sensitivity. Arylamidase is found in biliary tract cells, and increases in serum with cholestasis. Cystyl aminopeptidase (EC 3.4.11.3), originating from syncytium cells, is also measured by this method, as is cytosol or soluble aminopeptidase (`true LAP', EC 3.4.11.1). Conversely, when leucinamide is used as a natural physiological substrate, only true LAP, in the form of cytosol or soluble aminopeptidase, is specifically measured. True LAP is concentrated in the lymphocyte [1].

In sera of normal individuals, arylamidase levels are high, but LAP levels are low. An increase in serum LAP level indicates its release from damaged cells containing large amounts of LAP in their cytosol fractions, or derivations from cells in which the synthesis of large amounts of LAP has been induced. Reports mentioning an increased serum LAP level in SLE have been scanty, perhaps due to the measuring method used hitherto.

The increased serum LAP found in our patients with active SLE is thought to have originated from activated or dying lymphocytes. To examine further whether elevations in serum LAP levels reflect the underlying evolution of active disease, we studied the relationships of LAP to laboratory indicators and to the SLE Disease Activity Index (SLEDAI) [2] in 46 consecutive patients with active SLE.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty-six consecutive patients with active SLE (42 women and four men; mean age 39.5±14.6 yr), who were admitted to the Tokyo Metropolitan Komagome Hospital from 1991 to 1996, were studied. The diagnosis of SLE was made by using revised ARA criteria [3]. They had been taking steroid (mean dosage 5.17±7.12 mg/day as prednisolone) at the time of admission. For a disease control group, LAP and C-reactive protein (CRP) levels were measured in 46 consecutively admitted patients with rheumatoid arthritis (37 women and nine men; mean age 62.8±11.1 yr).

The laboratory indicators measured and the normal ranges are listed in Table 1Go. The normal range had been obtained from 406 female and 128 male healthy individuals in our laboratory centre by the log-normal graph paper method and was from 2.5 to 97.5%. The level of {gamma}-globulin was calculated as the total protein concentration multiplied by the {gamma}-globulin fraction ratio. The data shown are those obtained when LAP levels peaked during their admission, from the blood samples taken at the same time. Serum LAP levels were measured by ULTRET LAP, obtained from Ono Pharmacy, Osaka, Japan. In this kit, the substrate used is L-leucinamide, and NADP-dependent glutamate dehydrogenase is linked, simultaneously detecting ammonia liberated by LAP.


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TABLE 1.  Laboratory findings and activity index, and their correlation with LAP level, in 46 patients with SLE admitted to Tokyo Metropolitan Komagome Hospital from 1991 to 1996
 
Eleven items listed in the ARA criteria [2] were checked to determine the involvement of any organ, in the disease process, in addition to liver involvement. Fever was considered to be present when the temperature exceeded 38.0°C. Disease activity was assessed by the SLEDAI. Data were analysed by using the correlation test, the {chi}2 test and Mann–Whitney U-test, with laboratory data in logarithmic form. Data are shown as the mean±S.D., unless otherwise indicated.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
LAP levels were measured 13.3±11.5 times during 100.5±71.2 days of admission. Thirty-three of the 46 patients had at least one result with elevated LAP levels. Five patients showed extremely high LAP levels, >1000 IU/l, and all of these five had high lactate dehydrogenase levels (>700 IU/l). A reduced lymphocyte count was seen in 31 patients. All five patients with extremely high LAP levels had lymphocyte counts <500/µl. LAP levels decreased to a mean lowest level of 39.8 ± 12.3 IU/l. The laboratory data, as well as SLEDAI scores obtained when LAP levels peaked, are shown in Table 1Go. The mean serum LAP level in the 46 patients with rheumatoid arthritis was 34.2±8.7 (from 15 to 50) IU/l (P<0.0001 by Mann–Whitney U-test, vs SLE patients). Their mean CRP level was 5.1±3.8 mg/dl.

The mean steroid dosage was found to be increased to 10.3±13.9 mg/day when LAP levels peaked. In 41 patients, no additional steroid had been administered before the LAP levels peaked; in four patients, LAP levels were increasing and reached peak levels just after steroid therapy was begun, and in one patient LAP levels increased even after steroid therapy was begun to treat a clinical exacerbation of the disease. The mean maximum dosage of peroral steroid was 21.0±21.9 mg/day. Five patients had i.v. pulsed steroid therapy. Two patients had cyclophosphamide pulse therapy after LAP levels had peaked.

The statistical correlations between LAP levels and the levels, activities and counts of other indicators and SLEDAI scores are shown in Table 1Go with P values by correlation test. Lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and {gamma}-glutamyl transpeptidase levels showed a highly significant, positive correlation with LAP. CRP, alkaline phosphatase and {gamma}-globulin levels showed no correlation with LAP levels. CH50 had a negative, and anti-DNA titres a positive, correlation with LAP levels. ß2-Microglobulin levels were distributed widely, with high levels in patients with active disease and in those with renal disorders, but showed no correlation with LAP levels. Lymphocyte and white blood cell counts, and neutrophil counts to a lesser degree, had a negative correlation with LAP levels. Platelet counts showed no correlation with LAP. There was a highly significant correlation between the SLEDAI scores and LAP levels.

For indicators listed in the ARA criteria, all cases were positive for ANA. Forty-three patients had haematological disorders (of whom 31 had high LAP levels), 33 had immunological disorders (25 with high LAP levels), 28 had arthritis (20), 19 had malar rashes (15), 18 had neurological disorders (14), 15 had renal disorders (9), nine had discoid rashes (6), eight had photosensitivity (6), four had serositis (3) and three had oral ulcers (2). The presence or absence of each of these disorders showed no correlation with elevated LAP levels by the {chi}2 test (data not shown).

An ultrasound scan of the liver was carried out in 24 patients with elevated LAP levels. Haemangioma was found in three patients, fatty liver in one, silent gall bladder stone or polyp in three, and transient thickening of gall bladder wall in one. Although four patients had hyper-{gamma}-globulinaemia >2.5 g/dl, none were considered likely to have autoimmune hepatitis. None had been given a diagnosis of viral hepatitis or alcoholic disorders. No patient had been on potentially hepatotoxic drugs, including cyclophosphamides or azathioprine, at least for a month before LAP peaked.

Fever was seen in 12 patients, was of no other obvious cause than SLE, and subsided without antibiotics. No patient had a diagnosis such as bacterial or viral infections, leukaemia, lymphoma or other malignancies. All 12 patients with a fever of over 38° C had elevated LAP levels (971.3±1043.3 vs 58.0±51.9 IU/I in patients without fever; 2.78±0.20 vs 1.70±0.04 in logarithm P<0.0001 by Mann–Whitney U-test).

All 46 patients left our hospital in a state of remission, with reduced LAP values.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, no diagnosis of active liver disease was made in any patient with elevated LAP levels. Indeed, it is unlikely that such a large number (>70%) of the patients with SLE would have liver disorders. The lack of correlation with alkaline phosphatase level agrees with the absence of liver or bile duct disorders. LAP levels correlated positively with lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase levels. Lactate dehydrogenase is distributed universally and heavily in most cells, does not vary in concentration among different kinds of cells, and is an indicator of the presence of enzymes derived from damaged cells. The latter two enzymes are also found in various cells. It is likely that LAP levels increased together with levels of these enzymes, without the presence of liver disease. Although lupus patients have been said to show occasional elevations of liver enzymes, a part of these findings might be actually with true LAP increase of lymphocyte origin.

Lymphocyte counts and white blood cell counts showed a strong negative correlation with LAP levels. Leucopenia and lymphocytopenia are important diagnostic criteria for SLE, often progressing in an active phase. Increased serum LAP and lactate dehydrogenase levels have been reported in measles and rubella, suggesting that both enzymes originated from T lymphocytes [4], which are thought to be directly infected in measles [5, 6]. T-cell mitogen was reported to increase intracellular LAP and lactate dehydrogenase levels, but B-cell mitogen did not [7, 8]. These results suggest that LAP originates from T cells that are active or destroyed during the active phase of SLE, although any direct evidence has not yet been obtained.

The enzyme {gamma}-glutamyl transpeptidase is membrane bound and functions in amino acid transport. It is located in epithelial cells, neutrophils, monocytes and lymphocytes. Normal human peripheral B cells have higher {gamma}-glutamyl transpeptidase activity as differentiation and maturation progress, as umbilical cord blood lymphocytes have lower activity, and lymphocytes in tonsil tissue have higher activity [10]. When erythrocyte rosette-forming cells were separated from the peripheral blood of patients with SLE, they showed increased {gamma}-glutamyl transpeptidase activity [11]. As {gamma}-glutamyl transpeptidase has been recognized as a characteristic B-cell marker, the correlation of its level with that of LAP suggests an activation of, or an interaction between, both cell lines in the active phase of SLE.

Accelerated apoptosis of lymphocytes isolated from patients with SLE has been reported in in vitro tissue culture [12], showing a significant correlation with the disease activity of SLE. Although no correlation of the level of ß2-microglobulin, which is concentrated in lymphocytes, with LAP level was found in this study, it may be because ß2-microglobulin also increases in cases of disturbed renal function.

Anti-DNA antibody titres showed a positive correlation, and CH50 showed a negative correlation, with LAP levels. Conversely, organ involvements experienced by the present patients varied from patient to patient; no common organ involvement was identified as being related to increased LAP. These data incline us to speculate that the higher the LAP level, the higher the likelihood of disease activity involving the lymphocyte immune system as the only common underlying pathogenic phase. The decrease in LAP level after recovery, seen in all 33 cases with elevated LAP levels, also supports this speculation.

We measured the SLEDAI score in these patients, and found a strong correlation with serum LAP levels. The SLEDAI is a popular means to evaluate disease activity, separating it from disease severity or the resulting damage. In the SLEDAI, each descriptor is weighted as 8, 4, 2 or 1, if present within the preceding 10 days [1]. Low complement and increased DNA binding carry a weight of 2 each, and leucopenia and fever have only 1 each. So the strong correlation between LAP level and either lymphocyte count, anti-DNA antibody levels, CH50 or fever do not seem to contribute much to the strong correlation between serum LAP levels and the SLEDAI score found in this study. Rather, this may suggest an underlying evolution of a pathogenic process involving lymphocytes. Also supporting this theory is the fact that LAP levels were almost within normal limits in patients with rheumatoid arthritis, in which those features described earlier are not characteristic.

In summary, slight to extreme elevations of LAP level were seen in >70% of patients admitted with SLE, when LAP levels were measured using leucinamide as the substrate. The excess LAP seemed to derive from activated or destroyed lymphocytes. From all of the clinical features of the patients with increased LAP levels, including fever, lymphopenia and leucopenia, increased anti-DNA antibodies, hypocomplementaemia, and the finding of decreased LAP levels during disease remission, it follows that an increase in serum LAP level may be an important indicator for active evolution of SLE. The strong correlation between LAP levels and the SLEDAI score supports this conclusion.


    Acknowledgments
 
The authors would like to thank Dr Victoria Elegant and Ms Keiko for their excellent help.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 29 April 1998; revised version accepted 5 March 1999.



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