Utility of plasma fluorometric emission scanning for diagnosis of the first 2 cases reports of variegate porphyria: a very rare type of porphyrias in Thai.

Two Thai women who are siblings presented with a history of recurrent pruritic vesicles on dorsum of both hands and extensor surface of forearms where the sun-exposed areas are. The excoriated vesicles were healed with depressed scars. They had no previous history of intense abdominal pain, seizure, or psychiatric disorder Urinary porphyrins were analyzed by High Performance Liquid Chromatography (HPLC). The level of coproporphyrin III was detected to be higher than the uroporphyrin level. Fluorescence emission scanning of both patients' plasma was performed and demonstrated typical emission peak at 626 nm, that confirmed the diagnosis of variegate porphyria.

Porphyrias diagnosed types by HPLC analysis: 10 years of Siriraj experience.

Objective: This study aims to report the HPLC patterns of urine porphyrin intermediates from Thai patients with various types of porphyrias in supporting the clinical diagnosis. Methods: A reverse phase HPLC method using kit reagents, was used to measure porphyrin intermediates in the urine of control subjects and patients with various types of porphyrias. Results: 22 control subjects showed very low levels of all urine porphyrin intermediates whereas 11 porphyriatic patients had increases of some specific isomers varying among each type of the disease. The results from 6 porphyria cutanea tarda (PCT) patients: marked increase of uroporphyrin and slight increase of the other porphyrin intermediates, 2 congenital erythropoietic porphyria (CEP), high elevation of uroporphyrin and coproporphyrin I – III ratio with slight increase of pentaporphyrin, 2 variegate porphyria (VP), marked increase of only coproporphyrin III and 1 acute intermittent porphyria (AIP) (non acute form), high increase of coproporphyrinIII with mild increase of ALA, PBG, uroporphyrin, and coproporphyrin I. Conclusion: The HPLC could provide data essential for differentiating common types of porphyrias in Thai patients, PCT, CEP, VP and AIP. Clinical findings of the patients and urine screening test for increased porphyrins were also helpful for the definite diagnosis.

Microalbuminuria analysis in Thai patients with diabetes and hypertension using albumin blue 580 fluorescence assay.

Objective: To evaluate the performance of the fluorescence assay using albumin blue 580 for microalbuminuria, which is one of the early signs of renal diseases and an important cardiovascular risk factor for patients with diabetes and hypertension. Methods: The fluorescence assay was tested for its precision and reliability by determining the intraassay and interassay coefficients of variation (CV). The correlation of the assay with the standard immunoturbidimetric assay (DCA 2000ฎ microalbumin/creatinine reagent kit), which is one of the methods routinely used for microalbuminuria, was evaluated by quantitating the urinary albumin levels in 13 urine samples by both methods and the results were compared. The fluorescence assay was also used to detect the presence of microalbuminuria in 11 healthy subject, 11 patients with hypertension, and 10 patients with diabetes and hypertension. Results: At the albumin concentrations of 5, 50, and 150 mg/L, the intraassay CVs of the fluorescence assay were 7.9, 4.4, and 3.5%, while the interassay CVs were 4.1, 8.0, and 0.4%, respectively. The fluorescence assay also showed a very good correlation with the standard immunoturbidimetric assay, with the intraclass correlation coefficient of 0.94 (0.81 to 0.98 at 95% confidence interval). When the assay was used to detect the presence of microalbuminuria (the excretion of 30-300 ตg albumin/mg creatinine), it identified two out of 11 patients with hypertension (18%) and three out of 10 patients with both diabetes and hypertension (30%) having microalbuminuria whereas none of the healthy subjects had the condition. In addition, the presence of clinical albuminuria (the excretion of more than 300 ตg albumin/mg creatinine) could also be identified in three patients with hypertension (27%) and one patient with both diabetes and hypertension (10%) respectively. Conclusion: The fluorescence assay using albumin blue 580 was found to be precise and reliable and also showed a very good correlation with the standard immunoturbidimetric assay. In addition, the fluorescence assay is simple and the assay cost is much cheaper compared with the immunoturbidimetric measurement. Therefore, it could be another alternative method for microalbuminuria, particularly for most hypertensive or diabetic patients in Thailand, who can benefit from the detection of microalbuminuria but cannot afford regular tests.

Comparative analysis of serum ceruloplasmin levels in wilson disease by conventional enzymatic assay and immunologic method.

Objective: Serum ceruloplasmin, which has markedly decreased in 95% of patients with Wilson disease, is one of the most useful markers in the diagnosis of this genetic disease. The disease is caused by an impairment of the excretion of hepatic copper, resulting in toxic accumulation of the metal in the brain, liver and other organs. Definite diagnosis leads to the need of continual, lifelong and effective treatment. Therefore, the accuracy of the measurement of this serum protein is clinically needed. Our study is aimed to compare the reliability of the two methods used in measuring serum ceruloplasmin: the conventional enzymatic assay and the recent immunologic method by using kit reagents. Methods: Serum ceruloplasmin levels were performed by the conventional enzymatic assay as reported by Ravin in 1961, and compared to the immunologic method using kit reagents, Dade Behring Inc., Newark, USA. Seven patients with clinically proven Wilson disease and twenty-two controls were recruited for the study. Results: The mean  SD levels of serum ceruloplasmin from all patients and controls as measured by the enzymatic assay were 1.58  2.28 mg/dl and 28.94  9.60 mg/dl, respectively. The serum levels from those patients measured by the kit assay were less than 8 mg/dl while the mean  SD of controls were 25.91  7.71 mg/dl. All serum ceruloplasmin levels after measurement by both assays showed a strong correlation coefficient (r = 0.8713; p-value < 0.01), with a significant decrease in all patients with Wilson disease when compared to controls. Conclusion: Our study supported the high correlation between the conventional enzymatic assay and the recent immunologic method in measuring serum ceruloplasmin. Although the analysis kit is expensive, it is more advantageous for routine laboratory service because of its simpler, automated test with a well-accepted quality control.

Comparative study of two screening tests for urinary porphyrins in the diagnosis of porphyrias.

Objective: To evaluate the efficiency of two urinary porphyrins screening tests: routine fluorescent detection and semi quantitative spectrophotometric scanning. Methods: Minimal-level detection was performed by adding standard coproporphyrin of 0, 25, 50, 100, 250 and 500 ตg/L in urine and then screened by the two methods. Urine samples from 39 controls, 7 patients with porphyrias and 20 patients with liver impairment were quantitated for total porphyrins, followed by a comparison of the two qualitative tests. Results: The fluorescent test detected the minimal porphyrin level at 250 ตg/L whereas spectrophotometric scanning could detect a lower level, at 100 ตg/L. Total control subjects showed negative results from both tests while all 7 patients with porphyrias and 6 out of 20 cases of liver impairment showed definite positive results. Conclusion: Urinary screening for porphyrins from both tests revealed the same accuracy from this study. Still, the spectrophotometric method which is simpler, more sensitive and easily interpretable seemed more practical as a screening test in general laboratories. Keywords: Screening test; Porphyrias; Urinary porphyrins

A girl with A glowing tooth : A case of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria is a rare type of porphyria caused by inherited defects of uroporphyrinogen III synthase, an enzyme in the heme biosynthetic pathway. The resultant accumulation of porphyrins causes damage to the skin and erythrocytes, leading to cutaneous photosensitivity and hemolytic anemia. Furthermore, excess porphyrins are also deposited in tissues, bone, and teeth, resulting in a reddish-brown discoloration of the teeth (erythrodontia) which fluoresces under long-wavelength ultraviolet light. In this report, a case of a 9-month old infant girl with recurrent skin eruptions, anemia with hepatosplenomegaly, and erythrodontia is presented. The diagnosis of congenital erythropoietic porphyria was made based on the clinical manifestations and biochemical investigations. The patient was treated successfully with allogenic bone marrow transplantation and is still in remission after almost 3 years posttransplantation.

Cytotoxicity and apoptosis of ovarian and breast cancer cell lines induced by OVS1 monoclonal antibody and paclitaxel.

OVS1 monoclonal antibody (MAb) produced against ovarian cancer is currently used to identify mucinous cystadenocarcinoma antigen as a tumor marker secreted in serum. The potential of OVS1 MAb in ovarian cancer treatment was studied by evaluating the induction of cytotoxicity and apoptosis of SKOV3 ovarian cancer and BT549 breast cancer cell lines induced by OVS1. Paclitaxel, an antitumor drug, was used as positive control and applied as a combined drug together with OVS1 MAb. OVS1 MAb and paclitaxel were found by MTT assay to induce cytotoxicity against both cell lines. The ED50 of OVS1 MAb were 26.25 and 25.00 microg/ml and of paclitaxel were 21.88 and 9.20 nM against SKOV3 and BT549 cell lines, respectively. The quantitative amount of cells determined by fluorimetric assay was correlated to the results of the MTT assay. The combined application of OVS1 MAb and paclitaxel on these two cell lines resulted in a greater cytotoxicity than observed by either agent alone. OVS1 MAb and paclitaxel applied against both cell lines induced the morphological changes of apoptotic cell death at 24 hours visualized by two color fluorescence dyes, Ho33342 and propidium iodide. Combination of the two substances enhanced the rate of apoptosis compared to either OVS1 MAb or paclitaxel given alone. DNA fragmentation was detected in an agarose gel electrophoresis after treating cells with OVS1 MAb and paclitaxel at 24 hours. These findings on the induction of cytotoxicity and apoptosis by OVS1 MAb on cancer cell lines have implications on the potential application of OVS1 MAb for clinical therapy.

p53 tumor suppressor gene mutation in ovarian cancer in Thai patients.

OBJECTIVES: To characterize molecular mutations of p53 gene in Thai ovarian cancer and compare the mutations with their pathological and clinical findings. MATERIAL AND METHOD: Direct DNA sequencing of hot spot region of p53 gene (exons 5 to 8) from 28 primary ovarian cancer tissues, 2 metastatic tumors and their paired blood samples was performed. The detected mutations were compared to the pathological and clinical findings and responsiveness to treatments after 36 months of follow-up. RESULTS: One insertion and 4 point mutations in exon 5 of p53 gene were found in 5 out of 28 (18%) ovarian cancer patients. There was no mutation in the paired blood samples. The histological types of the detected tumors were 3 endometrioids and 2 serous cystadenocarcinomas. All 5 patients were in stage I to IV disease and showed overall 4 out of 5 (80%) complete response until 36 months after surgery followed by chemotherapy, compared to 14 out of 28 (50%) of complete response in all cases of ovarian cancer. CONCLUSION: The authors found 5 cases of ovarian cancer patients with p53 gene mutations giving the same response to complete standard treatment as all cases. Significant factors affecting responsiveness of these patients depended more on stages, grades and histological cell types of the cancer.

Comparative analysis of venous blood lactate levels by automatic lactate strip kit (accusport) and by standard marbach’s enzymatic method.

Lactic acidosis is an emerging life-threatening condition that needs to be diagnosed and treated as early as possible. The complete analysis of blood lactate levels by standard method takes at least a few hours and is not available at all times. The automatic lactate strip kit (Accusport) will be more practical for diagnosis and treatment of lactic acidosis patients. This study showed the results of blood lactate determined by standard enzymatic method of Marbach compared to the lactate strip. The results showed a strong correlation between the two methods (r2=0.966). The correlation increased in the case of high blood lactate levels (r2=0.978) and decreased in normal blood lactate levels (r2=0.943). Overall lactate values measured from the strip method were lower than those from the enzymatic method. From this study we can calculate a constant factor of 0.981 which when multiplied with the value of blood lactate analysed by lactate strip then added with 0.532, the result will be equal to that from Marbach’s enzymatic method.

Serum ceruloplasmin in wilson’s disease.

Wilson’s disease is a rare inherited disease with an incidence of 1:50,000 to 1:1,000,0001. The mode of transmission of this disease is an autosomal recessive. The defects of the disease involve both an impairment in biliary copper excretion and a decreased incorporation of free copper into ceruloplasmin due to a low level or abnormal function of this alpha 2-globulin. The toxic accumulation of copper damages many tissues especially basal ganglia of brain and liver. The diagnosis of this disease is made from the family history, signs and symptoms especially from the damaged organs, the detection of Kayser-Fleischer ring, a copper deposit in cornea and the decreased level of serum ceruloplasmin below 20 mg/ml which is normally found in more than 95% of cases. In this study, we analysed the levels of serum ceruloplasmin from 23 cases of Wilson’s disease admitted in Siriraj Hospital from 1989-1998. The average value, (X+1 SD), of 1.38+3.55 mg/ml was significantly lower when compared with the value of 20.82+3.63 mg/ml from 46 normal controls. The cut off level of ceruloplasmin for Wilson’s disease in Thai patients from this study was estimated to be 13 mg/ml which is lower than the level of 20 mg/ml in standard textbook. Twenty two out of 23 cases of Wilson’s disease (95.65%) had serum ceruloplasmin levels less than 13 mg/ml while 1 out of 23 cases (4.35%) had normal level. The data strongly supported the usefulness of serum cerloplasmin analysis in the diagnosis of Wilson’s disease.

Mitochondrial genetics of mitochondrial diseases in Thailand.

Mitochondrial diseases are a heterogenous group of disorders with various biochemical defects in the oxidative phosphorylation system. They are included in the differential diagnosis of many cases of multisystem disease even though the neuromuscular and central nervous system are mainly involved. The complexity of the disease can make it difficult for the clinician to diagnose. The number of the patients suffered from Mitochondrial disease is expected to be increasingly found each year. We present here the mitochondrial diseases with their underlying molecular genetics in the mitochondrial DNA found in Thai patients.

Establishment of OVS3 monoclonal antibody recognizing human ovarian cancer.

OVS3 monoclonal antibody (MAb) was established by fusing murine myeloma cell line NS 1/1-Ag 4-1 with spleen cells from mouse immunized with fresh ovarian endometrioid carcinoma. The MAb was selected by a specific immunohistological (streptavidin-biotin) staining on the tumor area of frozen sections of the same tumor. The immunohistological staining with OVS3 MAb was also screened on frozen sections of 21 normal tissues, 5 benign tumors, 20 ovarian cancers and 5 other cancers. The positive result was 50% in all ovarian cancers compared to almost negative results in the other groups (97% specificity). The heat labile property of OVS3 antigen (Ag) was found when hegative staining results were shown in all paraffin sections in spite of positive reactions on frozen sections of the same tumors. The OVS3 Ag was confirmed to be a 3-subunit protein of 40-50 kD molecular weight by the immunoprecipitation technique. The data supported OVS3 MAb as another new MAb recognizing an epitope different from the previously established OVS1 and OVS2 MAbs.

Evaluation of new ovarian cancer markers, STN, CA 546 and CA 72-4 in Thai patients.

Ovarian cancer is the most fatal gynaecologic malignance. At present, there is no sensitive test to detect early stages of the disease. In our study of 32 ovarian cancer patients admitted in Siriraj Hospital, 3 new tumor makers, STN, CA 546 and CA 72-4 were selected to evaluate for their sensitivity as compared with CA 125 Kit. The best positive rate for non-mucinous type of ovarian cancer was 82% obtained from the CA 125 test while the result for mucinous type was 67% obtained from a combined test of CA 125 and STN. As a requirement for early diagnosis, tests using CA 125 or CA 72-4 showed best sensitivity of 33% in early stages of non-mucinous, while CA 546 test revealed the highest result of 33% for mucinous type. Thus, the combination of new tumor marker assay of STN, CA 546, CA 72-4 together with CA 125 would increase sensitivity in detecting ovarian cancer especially for the mucinous type which is more common in Thailand.

The determination of beta-aminoisobutyric acid in urine of normal and cancer patients.

Beta-aminoisobutyric acid (βAIBA); a degradation product of thymine in both deoxyribonucleic (DNA) and transfer ribonucleic acid (tRNA) was determined in single urine specimens from 65 normal healthy subjects and 60 patients with cancer of various organs by thin layer chromatography. The normal control value from both males and females was 74.01+25.81 nmol βAIBA/µmol creatinine. The excretion levels were significantly increased in patients with several types of tumor (146.79+137.64 nmol βAIBA/µmol creatinine). Increased levels of βAIBA excretion may reflect changes in DNA metabolism or increased turnover of tRNA in tumor. The changes of βAIBA excretion levels were also studied in cancer patients before radiotherapy. The levels returned to normal when they were in remission but remained high in patients whose clinical conditions were not improved. The same results were also found in patients with choriocarcinoma during chemotherapy. Therefore, determination of urinary βAIBA level could be used as a potential biological marker for monitoring the effectiveness of therapy in malignancies.