QUARTERLY Monitoring of urine nitric oxide (NO) related substrates and immunological competence in hematological malignancy �

It has been reported that concentrations of neopterin in the urine are changed according to the host immunological conditions. In the present study, we measured urinary concentration of neopterin in patients with malignant hematological disorders and investigated the relationship between urinary neopterin levels and laboratory indices for cellular immunity. Urine neopterin levels were correlated with serum sIL-2R levels in the patients with malignant lymphoma, and inversely correlated with lymphocyte reactivity with ConA in the patients with acute myelocytic leukemia. However, no significant correlation was observed between urine neopterin levels and lymphocyte reactivity with phytohemagglutinin and pokeweed mitogen, CD4/8 ratio, CD56+ 16+ subset or serum IFN-gamma levels. In the patients with malignant lymphoma, parallel changes in serum sIL-2R and urine neopterin were observed. The presented results suggest that urine neopterin levels are related to the activation of T cells in malignant lymphoma.

Recently, it has been revealed that nitric oxide (NO) is produced by T cells or macrophages [1][2][3], suggesting that NO is associated with cellular immunity.Tetrahydrobiopterin (BH 4 ), a cofactor for NO synthase (NOS), is produced when the cells involved in cellular immunity are activated [4,5].Furthermore, it has been reported that urine concentrations of neopterin, an intermediate product of BH 4 , changes according to immunological conditions of the host [6,7].These results suggest that NO and BH 4 are involved in cellular immunity.In the present study, we measured urinary concentrations of neopterin in patients with malignant hematological disorders and investigated the relationship between urinary neopterin levels and laboratory indices for cellular immunity.

EXPERIMENTAL PROCEDURES
Urine samples for neopterin (NP) assay were collected from two patients with malignant lymphoma (ML), three patients with acute myelocytic leukemia (AML) and two patients with multiple myeloma (MM) who were hospitalized in Shimane Medical University Hospital.Among these patients, autoperipheral blood stem cells transplantation was carried out in Cases 1 and 6.Urine samples were collected at the time of admission, pre-and post-chemotherapy, and pre-and post-transplantation.To avoid the influence of inflammation, urine samples were not collected when patients were assumed to have inflammation as diagnosed by fever and increased serum C-reactive protein.Urine sample was centrifuged at 3000 r.p.m. for 10 min.The supernatant was stored at -80°C until assayed.Urinary neopterin levels were measured according to the method of Hibiya et al. [8].Briefly, an aliqout of the urine supernatant (120 ml) was added to 60 ml of 6 M HCl and boiled at 100°C for 2 h.Then the sample was lyophilized and reconstituted with 120 ml of 50 mM ammonium phosphate (pH 3.0).The mixture was centrifuged at 4 200 r.p.m. for 10 min.The supernatant was applied on HPLC with fluorescent detection (excitation: 350 nm, emission: 455 nm).As indices for cellular immunity, lymphocyte reaction with phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) were estimated using JIMRO Fluorometric Blastformation Test (Japan Immunoresearch Laboratories Co., Ltd., Takasaki, Japan), that is measured amount of nucleic acid of reacted lymphocyte by lectin (PHA, ConA and PWM).CD4/8 ratio and CD56 + 16 + cell subset were measured using flow cytometry.Soluble IL2 receptor (sIL2R) levels in the serum measured in SRL Inc. Laboratory.Serum interferon g (IFN-g) levels were estimated using ELISA assay kit (Amersham Pharmacia Biotech, Tokyo, Japan).

RESULTS
Immunological indices and urine NP levels of the patients are shown in Table 1.Serum sIL-2R levels of the patients with ML were much higher than those of the patients with AML and MM, and beyond the healthy control estimated in the Laboratory.Other immunological indices were within healthy control range and not different among the patients with ML, AML and MM.Urine NP levels did not differ among the patients with ML, AML and MM.Urine NP levels were correlated with serum sIL-2R levels in the patients with ML, but not AML or MM (Fig. 1).Regardless of the disease, urine NP levels varied markedly between samples from the same patient.The NP levels were inversely correlated with lymphocyte reactivity with ConA in the patients with AML (Fig. 2).No significant correlation was observed between urine NP levels and lymphocyte reactivity with PHA and PWM, CD4/8 ratio, CD56 + 16 + subset or IFN-g levels.In the patients with ML, urine NP levels varied parallel to the serum sIL-2R levels (Fig. 3).In contrast, no relationship was observed between the profile of urine NP levels and lymphocyte reaction with ConA in the patients with AML.

DISCUSSION
Urine NP levels were remarkably elevated at some collection points in many patients beyond the healthy control levels [9].Since NP is altered by immunological disorders [6,7], it is the possibility that urine NP can be an index for T cell activation in the patients with ML.
In the patients with AML and MM, no correlation was observed between urine NP and se-   rum sIL-2R.In these patients, sIL-2R levels varied within the normal range.Thus it is assumed that urine NP levels are not as sensitive as the level of sIL-2R to T cell activation.The lymphocyte reaction with ConA is also a marker for T cell activation.From our data, the inverse correlation between urine NP and lymphocyte reaction with ConA in the patients with AML cannot be easily explained.Since no relationship was observed between the profile of urine NP and lymphocyte reaction with ConA in the patients with AML, urine NP is possibly affected by some factors other than T cell activation in AML.Taken together, these results suggest that urine NP level is correlated with the activation of T cells in some hematological disorders.

Figure 3 .
Figure 3. Profile of urine neopterin and serum sIL-2R in patients with multiple myeloma (ML).A. Case 1: a, after 2 courses of chemotherapy and recovery from bone marrow suppression; b, after 3 courses of chemotherapy and recovery from bone marrow suppression; c, after 4 courses of chemotherapy and bone marrow suppression state; d, after high dose of chemotherapy and before PBSCC; e, before PBSCT; f, after PBSCT; g, after PBSCT and recovery from bone marrow suppression; h, follow up.B. Case 2: a, after chemotherapy and recovery from bone marrow suppression; b, after chemotherapy and recovery from bone marrow suppression; c, after chemotherapy and recovery from bone marrow suppression; d, terminal.