Clinical evaluation of serum neuron-specific enolase levels in patients with lung cancer

Serum neuron-specific enolase (NSE) levels were studied in 105 patients with malignant neoplasms (lung cancer 38, others 67), 13 patients with various benign diseases and 7 healthy adults. The mean serum NSE level in adult control subjects was 7.4 +/- 0.8 ng/ml, and cut off level was decided 10 ng/ml. Serum NSE levels were elevated in 14/38 (37%) of patients with lung cancer and in 14/67 (21%) of patients with the other malignant neoplasms. In patients with benign diseases, serum NSE level was elevated only in one patient with pituitary adenoma. In 7 patients with small cell lung cancer, the positive rate was higher (86%) than in those with non-small cell lung cancer (26%), and serum NSE levels were higher than 25 ng/ml except one case. There was no correlation between serum NSE and CEA (carcinoembryonic antigen) levels in patients with small cell lung cancer, also in patients with lung cancer. The measurement of serum NSE level seemed to be useful for diagnosis in patients with small cell lung cancer.     

A fundamental and clinical study of ferritin 'Eiken' radioimmunoassay kit

We have measured serum ferritin level using double antibody radioimmunoassay kit (Eiken ICL) and evaluated the characteristics of the kit and clinical usefulness. Satisfactory results were observed in standard curve, reproducibility, dilution and recovery test. In clinical evaluation, we have measured in normal subjects and patients with various diseases. The range in normal males and females were 13.0-158.7 ng/ml and 7.3-73.0 ng/ml, respectively. Serum ferritin level was elevated in patients with hepatoma, biliary cancer, lung cancer and other malignant diseases. Measurement of serum ferritin value would be useful in the monitoring of cancer patients.     

Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 +/- 0.099 vs 0.130 +/- 0.020 mumol/g; CySSCy, 0.051 +/- 0.027 vs 0.013 +/- 0.002 mumol/g). Administration of N-nitrosobis(2-oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 +/- 0.272 vs 0.062 +/- 0.017 mumol/g; GSSG, 0.155 +/- 0.063 vs 0.011 +/- 0.001 mumol/g).     

Novel fluoroimmunoassay for ovarian cancer biomarker CA-125

Cancer antigen 125 (CA-125) is a glycoprotein biomarker that denotes the presence of ovarian and reproductive cancers in women, with serum concentrations of CA-125 greater than 35 U/ml considered indicative of potential malignancies. A fluorescent immunoassay recently developed in our laboratory employing the ALYGNSA antibody-orientation system has been used to measure CA-125 levels. This system displayed significantly increased sensitivity with a detection limit of 1.5 U/ml compared to that of a commercial CA-125 enzyme-linked immunosorbent assay (15 U/ml) This tenfold lower level of detection of the ALYGNSA CA-125 assay should permit better identification and monitoring of ovarian cancer.     

Determination of pioglitazone in dog serum using solid-phase extraction and high-performance liquid chromatography with ultraviolet (229 nm) detection

An analytical method is described for the determination of the free base of pioglitazone hydrochloride (U72, 107A, AD-4833) in dog serum. The method used solid-phase extraction of pioglitazone from serum followed by high-performance liquid chromatographic analysis on an octadecylsilane column with an eluent of acetonitrile-water (41:59, v/v) containing 1.2 ml/l acetic acid (pH 6.0 +/- 0.05). The column effluent was monitored at 229 nm. The analytical procedure has a linear range of 25 ng/ml to 20 micrograms/ml, a minimum quantifiable level of 25 ng/ml, absolute recovery of greater than 90% (n = 15), and precision of less than or equal to 8.8% (n = 45). The method was used in a preliminary dose proportionality study in the dog.     

Conjugated 1 beta-hydroxycholic acid in the urine of newborns and pregnant women measured by radioimmunoassay using antisera raised against N-(1 beta-hydroxycholyl)-2-aminopropionic acid-bovine serum albumin conjugate.

Anti-tauro 1 beta-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1 beta,3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oyl)-2- aminopropionic acid-bovine serum albumin (BSA) conjugate. The antisera raised had high affinity (1.25-1.46 x 10(9) M-1) and specificity for conjugated 1 beta-hydroxycholic acid; cross-reactivity for glyco 1 beta-hydroxycholic acid was 100% and that for the glycine and taurine conjugates of other 1 beta-hydroxylated bile acids ranged from 11.30 to 0.23%. Urinary concentrations of conjugated 1 beta-hydroxycholic acid were determined by radioimmunoassay in newborns, 0-20 d after birth, in amounts ranging from 0.29 to 18.51 micrograms/ml and in women in late pregnancy (less than 1.13 micrograms/ml), as well as in normal women (less than 0.12 microgram/ml).     

Immunochemical measurement and immunohistochemical detection of membrane-associated placental tissue protein 1

Using the avidin-biotin binding system, an enzyme immunoassay procedure was developed to measure the membrane-associated placental tissue protein 1 (MP1) in serum. The standard curve covered the range from 10 to 1000 ng/ml of MP1. The intra- and inter-assay coefficient of variations (C.Vs) were less than 5 and 10%, respectively. Recoveries of MP1 added to serum ranged between about 96 and 101%. The MP1 serum level was over 10 and under 112 ng/ml in non-pathological men, and under 240 ng/ml in non-pathological women. The MP1 level in the ovulatory phase was higher than in other phases of the menstrual cycle. In pregnancies during 6-39 weeks, the MP1 level ranged from 10 to 540 ng/ml, and it increased during the third trimester of gestational age. In benign gynecologic diseases, the MP1 concentration in serum ranged from 10 to 215 ng/ml. The MP1 levels in benign diseases were compared with those in ovarian malignancies, in endometrial carcinoma, and in uterine cervical cancer. The immunohistochemical location of MP1 was detected in the cell membrane of ovarian cystadenocarcinoma.     

Spectrofluorimetric determination of guanethidine sulphate, guanoxan sulphate and amiloride hydrochloride in tablets and in biological fluids using 9,10-phenanthraquinone

A highly sensitive spectrofluorimetric method for the determination of some drugs of the monosubstituted guanidine derivatives in laboratory made tablets, in spiked human serum and in urine samples is presented. The method is based on the reaction of guanethidine sulphate (I), guanoxan sulphate (II) and amiloride hydrochloride (III) with 9,10-phenanthraquinone (IV) to give highly fluorescent derivatives. The linearity ranges were found to be 0.06-0.96 mug/ml for (I) and (II) and 0.04-0.28 mug/ml for (III), with relative standard deviation less than 2%. Mean percentage recoveries for tablets were found to be 99.9 +/- 1.3, 100.5 +/- 1.1 and 100.0 +/- 1.6 for I, II and III, respectively. For I and III the results are highly correlated with the B.P. methods. Using the synchronous fluorimetry, differentiation between I and II was possible. Chloroform, dichloromethane and ethyl acetate have been used to extract I, II and III, respectively from serum and urine at basic pH, followed by applying the proposed fluorimetric method. Percentage recoveries were found to be 95.7-102.2%. The limit of detection is 0.04 mug/ml for I and II and 0.02 mug/ml for III.     

Fluorimetric determination of the levels of urinary neopterin and serum thiobarbituric acid reactive substances in the nonagenarians

Twelve self-sustaining nonagenarians, 10 women and two men, aged 94+/-3 years, and eight institutionalised nonagenarians, eight women, aged 91+/-1 year as well as 11 control subjects, seven women and four men, aged 84+/-5 years entered the study. Urinary neopterin, an indicator of systemic immune activation, and serum thiobarbituric acid reactive substances (TBARS), a marker of lipoperoxidation, were determined initially, and collection of the blood and urine samples was repeated at 3-month interval. Neopterin was measured in the urine specimens by reversed-phase high performance liquid chromatography. A C(18) reversed-phase column 3.3x150 mm, 5 mum-diameter packing Separon SGX was used. Potassium phosphate buffer (15 mmol l(-1), pH 6.4) at flow rate of 0.8 ml min(-1) was used as mobile phase. After centrifugation (5 min, 1300xg) and diluting 100 mul of urine specimens with 1.0 ml of mobile phase containing 2 g of disodium-EDTA per litre, a 20 mul sample was injected on a column. Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission). Creatinine was determined by Jaffé kinetic reaction after dilution of sample 1:50 (v/v). The concentration of neopterin in urine was expressed as neopterin/creatinine ratio (mumol mol(-1) creatinine). TBARS were determined spectrofluorometrically using LS-5 spectrofluorimeter (excitation wavelength 528 nm, emission wavelength 558 nm) after extraction with n-butanol treatment with thiobarbituric acid. The significance of differences between nonagenarians and control group was examined by ANOVA-Kruskal-Wallis tests, using statistical software NCSS 6.0.21 (Kaysville, UT, 1996). The decision on significance was based on P=0.05. Urinary neopterin was significantly higher in institutionalised compared to self-sustaining subjects and controls (625+/-565 vs. 203+/-63 mumol mol(-1) creatinine, and 198+/-128 mumol mol(-1) creatinine, respectively, P=0.006). The serum TBARS were higher in both groups of nonagenarians (3.23+/-1.16 mumol l(-1) and 2.69+/-0.39 vs. 2.12+/-0.83 mumol l(-1) for the self-sustaining, institutionalised and controls, respectively, P=0.023). We conclude that the fluorimetric determinations of urinary neopterin and serum TBARS can be useful for the monitoring health status in the elderly patients.     

Myocardial injury occurs earlier than myocardial inflammation in acute experimental viral myocarditis

Endomyocardial biopsy often fails to show myocardial inflammation for patients with clinically suspected myocarditis. The serum isoforms of troponin T (cTnT) level is a very sensitive marker of myocardial injury and it is elevated even in the absence of myocardial inflammation. We investigated the correlations for myocardial injury, virus titers and inflammation in acute viral infection. Using the murine coxsackievirus group B3 (CVB3) myocarditis model, the histopathologic findings and virus titers in mouse hearts were compared with the serum cTnT levels measured by ELISA at various time points. Viable virus titers in the hearts peaked at 3 days after infection (8.22 +/- 0.13 log10 PFU/100 mg of heart); they decreased at day 7 and no viable virus was detected from day 14. Myocardial inflammation was minimal at day 3, peaked at day 7 and markedly decreased at day 14. The individual serum TnT levels were significantly increased at day 3 (7.37 +/- 1.46 ng/ml), persisted to day 7 (0.73 +/- 0.08 ng/ml), and normalized at day 14. Serum cTnT levels were correlatable with virus titers in the heart (r = 0.744, P <0.01), but the serum cTnT levels were not correlated with the degrees of inflammation. Using the less myocarditic strain of CVB3, similar relationships were observed between the changes for the serum cTnT levels and the heart virus titers. During the course of viral infection, myocardial injury precedes the pathologic evidence of inflammation, and the elevated cTnT levels provide evidence of myocardial injury even in the absence of any histologic findings of myocarditis.     

血清CEA、CA19-9检测诊断老年胃癌的价值探讨

目的对应用血清CEA、CA19-9联合检测方式对患有胃癌疾病的老年患者的病情实施诊断的临床价值进行研究。方法选择广丰县中医院收治的患有胃癌疾病和胃部良性疾病的老年患者各60例,再抽取同期接受健康体检的健康老年人资料60例,分别将其定义为研究1组、研究2组、对照组。采用化学发光法对三组研究对象的血清CEA、CA19-9水平进行测定,对比分析检测结果和两项指标的阳性率。结果研究1组的血清CEA、CA19-9水平明显高于对照组和研究2组,组间数据比较差异显著(P0.05);研究2组的血清CEA、CA19-9水平明显高于对照组,组间数据比较差异显著(P0.05)。研究1组的CEA、CA19-9水平检测阳性人数明显多于对照组和研究2组,组间数据比较差异显著(P0.05);研究2组的CEA、CA19-9水平检测阳性人数明显多于对照组,组间数据比较差异显著(P0.05)。结论患有胃癌疾病的老年患者的血清CEA、CA19-9水平会异常升高,临床上可以将其作为老年胃癌疾病诊断的重要依据。     

Clinical evaluation of immunoglobulin-E radioimmunoassay kit

An IgE RIA kit (Sandwich method; Dainabott), is used to obtain the following results. (1) Standard curve: Since the range of reproduction rate show 3.16-7.07% (C.V.), the curve become steep. (2) Incubations under controlled situation: Both of the incubations are controlled at 15-30 degrees C for 2 h. (3) Reproducibility test: Coefficients of variation (C.V.) of intra-assay and inter-assay variation are 2.32-3.94% and 2.92-3.92% respectively. (4) Recovery test: A result of the recovery test range between 100.1-101.7%. (5) Dilution test: Multiple dilution effects are observed. (6) Average counts of the serum IgE for the controlled and diseased groups: The average counts of the serum IgE for the controlled group, atopic diseased group, allergic rhinitis group and allergic bronchial asthma are 144.9 +/- 183.2 IU/ml, 1,099.0 +/- 2,782.4 IU/ml, 1,150.9 +/- 2,063.3 IU/ml and 600.7 +/- 686.4 IU/ml respectively. The value of the diseased groups have tendency to show higher averages than the controlled group. Since the controlled and diseased groups show wide distributions of the serum IgE level, there is no significant difference of two variations. However the diseased groups have tendency to show higher ratio of the serum IgE level in blood than the controlled groups. These basic researches are quite meaningful, because they are able to apply for a supplemental diagnosis of the atopic and parasitic disease.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women

Homocysteine (Hcy) is thought to play an important role in the development of osteoporosis and fracture. Methionine synthase reductase (MTRR) is an enzyme involved in the conversion of Hcy to methionine. We hypothesized that certain genetic polymorphisms of MTRR leading to reduced enzyme activity may cause hyperhomocysteinemia and affect bone metabolism. We therefore examined the associations of the A66G and C524T polymorphisms of the MTRR gene with bone mineral density (BMD) and serum osteocalcin levels in postmenopausal women. Although we did not detect any significant associations between MTRR polymorphisms and BMD or serum osteocalcin levels, we found that the 66G/524C haplotype, which has reduced enzyme activity, was significantly associated with serum osteocalcin levels in a gene-dose dependent manner (P = 0.002). That is, the highest osteocalcin levels (34.5 +/- 16.8 ng/ml) were observed in subjects bearing two copies, intermediate osteocalcin levels (32.6 +/- 14.4 ng/ml) were observed in subjects bearing one copy, and the lowest levels of osteocalcin (28.8 +/- 10.9 ng/ml) were observed in subjects bearing no copies. These results suggest that the 66G/524C haplotype of the MTRR gene affect bone turn over rate.     

Determination of total arsenic in serum and packed cellsof patients with renal insufficiency

In order to investigate the arsenic level in serum and packed cells of patients with renal insufficiency, total arsenic (As) concentrations were determined with hydride generation atomic absorption spectrometry (HGAAS) in serum (S) and packed cells (PC) of 31 non-dialyzed patients. The accuracy of the method was tested by the analysis of arsenic in 3 certified reference materials. Patients showed a three-fold increase of arsenic concentrations in serum and a two-fold increase of arsenic in packed cells compared with controls. Patients (n=10) with higher serum creatinine (>2.0 mg/dL), urea (>0.70 g/L) and urinary protein (mean+/-SD: 1.12+/-0.82 g/L) showed higher arsenic concentrations (5.8+/-3.3 microg/L in serum and 18.0+/-16.7 microg/kg in packed cells) compared with those with lower creatinine (<1.6 mg/dL), urea (<0.6 g/L) and urinary protein (mean+/-SD: 0.27+/-0.82 g/L) (n=16, serum arsenic 1.2+/-1.2 microg/L, packed cells arsenic 2.6+/-1.9 microg/kg). The significant differences (both p < 0.001) in S and PC-arsenic levels of patients in group I and II implies a relationship between the arsenic level and the degree of chronic renal insufficiency.     

Rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum.

A rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum has been developed. After deproteinization of the serum with 10% trichloroacetic acid, the samples were separated on a reversed-phase column, and subsequently reduced to their radicals with alkaline sodium hydrosulfite solution. These radicals were monitored with a UV detector at 391 nm. This method permitted the reliable quantification of paraquat over linear ranges of 50 ng - 10 microg/ml and 100 ng - 10 microg/ml for diquat in human serum. The within- and between-day variations are lower than 2.3 and 2.2%, respectively. This technique was also utilized to determine the paraquat and diquat serum levels in a patient who had ingested herbicide (Prigrox L) containing paraquat and diquat.     

Sensitive fluorimetric method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles. The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 +/- 3.1, and spermine 8.4 +/- 2.8 nmoles/ml packed erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml. The polyamine content of the erythrocytes from patients with malignant neoplasms was significantly elevated.     

Determination of ceftazidime in dolphin serum by liquid chromatography with ultraviolet-visible detection and confirmation by thermospray liquid chromatography-mass spectrometry.

A simple and sensitive liquid chromatographic method has been developed for the determination of therapeutic levels of ceftazidime in dolphin serum. The method involved an ultrafiltration of diluted serum with an equal amount of acetonitrile-ethanol-water (40:40:20, v/v/v) through a 10,000 daltons molecular mass cut-off filter. Separation of ceftazidime from the other serum components was performed by ion-paired (dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile-water solution. The ultraviolet absorbance of the column effluent was monitored in the 200-340 nm range of a photodiode-array detector or at 258.8 nm on a variable-wavelength ultraviolet-visible detector. Recoveries of ceftazidime from dolphin serum spiked with 20 and 2 micrograms/ml were 92.9 and 91.1% with coefficients of variation of 5.5 and 5.7%, respectively. A correlation coefficient of 0.9994 occurred with ceftazidime in aqueous solutions (n = 6, in duplicates). The limit of detection for this antibiotic was estimated to be approximately 50 ppb (ng/ml). The unbound ceftazidime concentrations in dosed dolphin serum were determined to calculate the protein bindings of this antibiotic which yielded 32 +/- 2%. The ceftazidime peak identity in dosed dolphin serum was confirmed by thermospray liquid chromatography-mass spectrometry. The thermospray mass spectrum of ceftazidime exhibited only the fragment ions, involving the opening of the beta-lactam ring, at m/z 237, 255 and 315 when positive-ion detection mode was employed and the fragment ions at m/z 235, 253 and 313 when negative-ion detection mode was used.     

Determination of pyridoxal 5'-phosphate in human serum by reversed phase high performance liquid chromatography combined with spectrofluorimetric detection of 4-pyridoxic acid 5'-phosphate as a derivative

A very sensitive spectrofluorimetric method for the determination of pyridoxal 5'-phosphate (PLP) in human serum is described. The specificity is based on the selective oxidation of PLP to 4-pyridoxic acid 5'-phosphate with potassium cyanide. Separation of the highly fluorescent 4-pyridoxic acid 5'-phosphate is achieved by reversed-phase high-performance liquid chromatography. Specificity is improved by a careful choice of fluorescence filters, maximised at the excitation (325 nm) and emission (418 nm) wavelengths of 4-pyridoxic acid 5'-phosphate. The detection limit for the reaction is 0.22 ng ml(-1). For quantification, the serum is spiked with PLP before protein precipitation with 3.3% m/V trichloroacetic acid. The method can be used for the determination of PLP in serum, even in vitamin B6 deficient patients. The mean value for human serum PLP from 30 healthy adults was found to be 14.6 +/- 4.8 ng ml(-1) (mean +/- standard deviation).     

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines

Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.