Growth and DNA synthesis of folate- and methionine-depleted L1210 mouse leukemia cells in culture.

The growth and DNA synthesis of L1210 mouse leukemia cells were examined under folate- and methionine-deficient conditions. Cell proliferation was dependent on methionine supplementation rather than on folate concentration. The UdR suppression value was abnormally high in the folate-deficient condition. However, it was also high when the methionine was low, despite folate supplementation. In accordance with this, UdR incorporation was significantly improved with various folates by cells grown in low-methionine conditions. Methionine depletion resulted in marked impairment of UdR incorporation regardless of folate concentration. These findings indicate close metabolic interrelations between folate and methionine, which may be relevant to the pathological biochemistry of human megaloblastic anemia.

[Significance of nucleic acid extraction for the performance of COBAS AMPLICOR Mycobacterium-PCR assay].

On an AMPLICOR-PCR assay for the diagnosis of mycobacterial infections, it has been found that approximately 10% of the routinely performed assays appear as indefinable cases with internal control (IC)-negative, which is caused by contaminant PCR-inhibitory substances in the extracted nucleic sample. To decrease the number of indefinable cases, we studied the IC performance of four nucleic acid extraction methods in one manual method(A) and three commercial kits(B, C and D). Thirty samples from 10 kinds of specimens without mycobacterial infections were subjected to the four nucleic extraction methods and the nucleic extracts were compared on PCR-inhibitory. The frequency of IC-negative samples was different among the four methods and the rates were 33.3%(n = 10) in A, 30.0%(n = 9) in B, 16.7%(n = 5) in C and 6.7%(n = 2) in D. In the PCR-inhibitory experiments using serially diluted IC-negative samples, the recovery of IC positivity was also different according to the method used. Although the IC-negative nucleic samples by D method easily became IC-positive at the 1/5 range of dilution, some of the IC-negative nucleic samples by A, B or C method were still IC-negative in the 1/10 range. These results indicate that the occurrence rate of indefinable cases with IC-negative is markedly different in the nucleic extraction method, suggesting that utilizing a suitable nucleic acid extraction method is important when using the diagnostic AMPLICOR-PCR assay.

[Pernicious anemia with indicanuria].

A 67-year-old female was diagnosed as having classical pernicious anemia. Laboratory data included low serum vitamin B12 concentrations, abnormal deoxyuridine suppression test, methylmalonic aciduria, atrophic gastritis, positive anti-intrinsic factor antibody and Schilling test results typical of pernicious anemia. During hospitalization it was incidentally noted that her urine was green colored. Jaffe' reaction and Obermayer reaction for indicanuria were both positive. Dark purple crystalline material was obtained by centrifugation of her urine. The crystalline substance was soluble in methanol and its absorbance curve was identical to that of authentic indoxylsulphate potassium salt. Daily output of this substance was nearly 50 times normal. There was no increase in urinary excretion of monoamino-monocarboxyl amino acides. The exact reason for her indicanuria was not clear, although abnormal bacterial growth in the intestine remained as a possibility.

[Urinary methylmalonic acid excretion and clinical features in megaloblastic anemia due to vitamin B12 deficiency].

Urinary methylmalonic acid (MMA) excretion in megaloblastic anemia due to vitamin B12 (B12) deficiency was studied using a colorimetric method. Average MMA excretion in 20 patients with untreated B12 deficiency was 164 mg/day, whereas it increased to 518 mg/day following oral administration of 10 g L-valine. Urinary MMA correlated significantly with platelet number, erythroblast percentage and deoxyuridine suppression test, while no correlation was found with hemoglobin, leukocyte number, reticulocyte, serum LDH, serum B12 and folate concentration. Patients with neurological disturbances excreted significantly larger amounts of MMA than those without neurological disorders. The results also indicated that MMA could be a useful adjunct for differentiation of megaloblastic anemia from myelodysplastic syndromes showing marked megaloblastic changes.

[Elderly non-hemophiliac patient with factor VIII inhibitor presenting various type hemorrhages].

A 81-year-old man who had been healthy without any history of abnormal bleeding, developed ecchymosis and hematuria in November, 1992 and was hospitalized in December, 1992. On admission, he developed widespread ecchymosis in his trunk and extremities, and subsequently ecchymosis of his cheek and neck, and also oral and pharyngeal hematoma. The laboratory data were as follows: whole blood clotting time, > 20 minutes; activated partial thromboplastin time (APTT), 108.6 seconds; Factor VIII activity, 4%. The level of Factor VIII inhibitor was high, 65.0 Bethesda Unit/ml. This inhibitor was a IgG type immunoglobulin, which had both kappa and lambda light chain. His serological and blood biochemical data of the blood were normal, and tests for autoantibodies were negative. The patient was treated with plasma exchange therapy, Prednisolone (PSL), Cyclophosphamide and Factor VIII concentrate. The hemorrhagic symptoms were improved, the inhibitor disappeared and the activity of Factor VIII returned to normal after one month. Follow-up was continued in the outpatient clinic for 5 months. After the dose of PSL was decreased, he developed bloody sputum and hematuria, and was readmitted in August, 1994. Factor VIII activity was 21% and the titer of Factor VIII inhibitor was 3.0 BU/ml. The hemorrhagic symptoms disappeared soon after increasing the dose of PSL, and the Factor VIII activity was normalized and the inhibitor could not be detected. These treatments appeared to offer effective control on severe hemorrhage in a patient with Factor VIII inhibitor.

Ki-1-positive large-cell anaplastic lymphoma with protean manifestations including central nervous system involvement.

A 26-year-old female with Ki-1-positive large-cell anaplastic lymphoma is reported. The neoplastic cells were phenotypically and genotypically of T cell origin. Initially, neoplastic cells invaded the skin and lymph nodes, and then invaded the sternal and vertebral bones, ribs and the iliopsoas muscle. Central nervous system (CNS) involvement with paraplegia, large tumor mass formation in her breast and pleural infiltration ensued in succession. This case illustrates the protean manifestations of Ki-1-positive large cell anaplastic lymphoma complicated with CNS involvement.

Hypercalcemia associated with osteolytic lesions in the extramedullary blastic crisis of chronic myelogenous leukemia: report of a case.

A 43-year-old male patient with hypercalcemia and osteolytic lesions complicating chronic myelogenous leukemia is presented. Extramedullary myeloid blastic crisis was diagnosed by the histological finding of the specimen biopsied from a osteolytic lesion in the right femur. As the serum levels of parathyroid hormone, 1,25 (OH)2 vitamin D, prostaglandin E2 and interleukin 1, and the urinary excretion of cyclic AMP were all normal, it was considered that the hypercalcemia was attributed to the bone destruction by the invasion of leukemic myeloblasts.

Increase in 3-deoxyglucosone levels in diabetic rat plasma. Specific in vivo determination of intermediate in advanced Maillard reaction.

A specific assay of 3-deoxyglucosone (3-DG) was developed in our laboratory to help elucidate the relationship between advanced Maillard reaction and diabetic complications. 3-DG is known as a highly reactive intermediate of the reaction in vitro and a precursor of advanced glycosylation end products such as pyrraline and pentosidine, which have been previously detected in vivo. 3-DG was converted to a stable compound, 2-(2,3,4-trihydroxybutyl)-benzo[g]quinoxaline, by reacting with 2,3-diaminonaphthalene. Since the derivative had a characteristic UV spectrum, it was determined at 268 nm by high performance liquid chromatography. This method was sensitive enough to detect 10 ng/ml (61.7 nM) of 3-DG in vitro. A slight modification to this method allowed in vivo detection of small amounts of 3-DG. Plasma free 3-DG levels were significantly higher in streptozotocin-induced diabetic rats compared with controls (918 +/- 134 nM versus 379 +/- 69 nM, p < 0.001) and were suppressed with the administration of aminoguanidine, an inhibitor of Maillard reaction. Plasma pyrraline levels in diabetic rats also increased in parallel with elevated 3-DG levels but were only marginally suppressed by administration of aminoguanidine. Our results indicate that 3-DG is present in vivo under normal conditions and that its level increases in diabetic subjects. Determination of 3-DG represents a good tool to predict development and progression of diabetic complications and to assess the efficiency of inhibitors to Maillard reaction.