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Phenylalanine and tyrosine quantification by stable isotope dilution liquid chromatography mass spectrometry from filter paper blood spots

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CLINICAL CHEMISTRY 1999年第4期45卷 571-573页

作者： Tuchman, M McCann, MT Univ Minnesota Dept Pediat Minneapolis MN 55455 USA

Phenylketonuria (PKU) is one of the more common inborn errors of metabolism, affecting 1 in 10 000–15 000 individuals (1). Newborn screening for PKU is performed in all 50 states and started with the development of the bacterial inhibition screening assay by Guthrie and Susi (2). Frequent monitoring of blood Phe concentrations is required to maintain the concentration as close as possible to the reference interval [60–120 μmol/L (1–2 mg/dL)] to prevent damage to the brain from increased Phe concentrations and to prevent a negative nitrogen balance from an overly Phe-restricted diet. Moreover, the high risk of birth defects for the fetus of a PKU mother requires extremely frequent monitoring of blood Phe concentrations and would greatly benefit from a rapid and accurate blood spot method. Phe determination from dried blood spots is feasible by various methods: bacterial (2), fluorometric (3), liquid chromatography (LC) (4), and tandem mass spectrometry (MS) (5). Of these methods, only tandem MS allows use of a stable isotope as internal standard. Although tandem MS is the most rapid method available, the extremely high equipment cost and the need for sample derivatization are disadvantages. We developed a rapid and precise blood-spot method for Phe and Tyr determination using stable isotope dilution reversed-phase LC-MS. The advantages of this method are the very short sample preparation without derivatization, a short chromatographic run, the use of selective detection to eliminate interference, and the use of ideal internal standardization (stable isotope). The method described herein is now used routinely in our PKU clinic.

关键词：血样采集色谱法高压液相质谱分析法纸苯丙氨酸/血液放射性同位素稀释技术酪氨酸/血液人类

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RADIOIMMUNOASSAY FOR DANAZOL IN HUMAN AND MONKEY PLASMA

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JOURNAL OF PHARMACEUTICAL SCIENCES 1978年第10期67卷 1425-1428页

作者： PETERSON, JE KING, ME BANKS, WF BAKER, JF JENSEN, AF ROSS, RW CLEMANS, S EDELSON, J STERLING WINTHROP RES INST DEPT DRUG METAB & DISPOSIT RENSSELAER NY 12144 USA

A sensitive method is described for the radioimmunoassay [RIA] of danazol in monkey and human plasma. Antiserum was developed in rabbits and a 2nd antibody was used to separate bound from free danazol. The RIA was specific for danazol and the limit of detection ranged from 1.4 to 2.8 ng/ml. Exogenous danazol could be quantitated accurately in both monkey and human plasma. The RIA results agreed with values obtained by inverse isotope dilution after i.v. administration of 14C-danazol to monkeys. The assay was used successfully to measure danazol in plasma from human volunteers receiving 200 mg of danazol.

关键词：达那唑/血液直鼻猴亚目猕猴方法孕二烯类/血液放射免疫测定放射性同位素稀释技术动物女(雌)性人类

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Assay for free and total carnitine in human plasma using tandem mass spectrometry

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CLINICAL CHEMISTRY 2000年第5期46卷 727-729页

作者： Stevens, RD Hillman, SL Worthy, S Sanders, D Millington, DS Duke Univ Med Ctr Res Triangle Pk NC 27709 USA

Measurements of free and total carnitine in plasma are important in the diagnosis and clinical management of patients with carnitine deficiency syndromes and certain inborn errors of metabolism (1). The most popular analytical methods are based on the enzymatic reaction in which an acetyl group is transferred from acetyl-CoA to carnitine with release of free CoA (2). A spectrophotometric assay based on this method, using a Cobas centrifugal analyzer (Roche Diagnostics), has been in use in this laboratory for the past several years (3). Limitations of the Cobas enzymatic method in our view include the initial filtration step, which requires a minimum sample volume of 300 μL, and the number and cost of the reagents required. Assay results are critically dependent on two labile reagents, the enzyme carnitine acetyltransferase and the color reagent 5,5'-dithiobis(2-nitrobenzoic acid). We have also observed that patients on valproic acid therapy sometimes give total carnitine values that are spuriously low for reasons that are not clear. Another limitation of the Cobas assay is that acylcarnitines with chain lengths >10 are lost in the filtration step (3), and the method will underestimate the total carnitine content of patients with defects in the oxidative degradation of long-chain or multiple acyl-CoAs, especially when metabolically decompensated. We have emphasized previously that the most valuable applications of the routine Cobas assay are the identification of patients with carnitine deficiency and the monitoring of patients on carnitine therapy (3).

关键词：乙酰肉碱/血液血蛋白质类/代谢肉碱/血液肉碱/代谢质谱分析法放射性同位素稀释技术氚人类

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COMPARISON OF RESULTS OF MICROBIOLOGIC AND RADIOISOTOPIC ASSAYS FOR SERUM VITAMIN-B12 DURING PREGNANCY

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AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY 1975年第1期122卷 21-24页

作者： GREEN, R COLMAN, N METZ, J S AFRICAN INST MED RES DEPT HAEMATOLPOB 1038JOHANNESBURG 2000SOUTH AFRICA UNIV WITWATERSRAND SCH PATHOLDEPT HAEMATOLJOHANNESBURGSOUTH AFRICA

The serum vitamin B12 level falls progressively during pregnancy, when measured either by microbiologic assay or by a radioisotope dilution technique. The concentration of the vitamin assayed by radioisotope dilution was consistently higher than the value obtained by microbiologic assay. This discrepancy was greatest in early pregnancy and decreased as the duration of pregnancy increased. These results add further evidence to the hypothesis that changes in serum binders for vitamin B12 are of major significance in the pathogenesis of the observed fall in serum levels of the vitamin during pregnancy.

关键词：生物学鉴定法血蛋白质类/代谢乳杆菌属/代谢妊娠妊娠并发症/诊断妊娠初期妊娠中期妊娠末期蛋白质结合放射性同位素稀释技术维生素B12/血液维生素B12/代谢维生素B12缺乏/诊断女(雌)性人类

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VALIDATION OF USE OF 11,12-H-2-LABELED CHENODEOXYCHOLIC ACID IN ISOTOPE-DILUTION MEASUREMENTS OF BILE-ACID KINETICS IN MAN

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PEDIATRIC RESEARCH 1975年第10期9卷 757-760页

作者： BALISTRERI, WF COWEN, AE HOFMANN, AF SZCZEPANIK, PA KLEIN, PD ARGONNE NATL LAB ARGONNEIL MAYO CLIN MAYO FDN GASTROENTEROL UNITROCHESTERMN 55901

Chenodeoxycholic acid labeled with 2H in the 11 and positions was prepared by catalytic reduction of delta 11-12 unsaturated derivatives of cholic acid. To validate the use of this stable isotope for the determination of bile acid kinetics by isotope dilution, it was administered to seven normal male volunteers simultaneously with [24-14C]chenodeoxycholic acid. Bile was collected at regular intervals over the following 5 days, and the chenodeoxycholic acid pool size and fractional turnover rate were determined from the specific activity decay curve for 14C and from the isotopic abundance curve for 2H. Estimates of the pool size by both isotopes showed a correlation of r = 0.95 and similar precision. Synthesis rate, the product of pool size and fractional turnover rate, also showed good agreement (r = 0.97), Because previous investigations have shown that bile acids tagged with hydrogen isotopes at the 11 and 12 positions are stable in man, the present data suggest that 11, 12-2H-labeled bile acids may be used in place of radioactive isotopes for valid isotopic measurement of bile acid kinetics in healthy infants and children.

关键词：碳放射性同位素鹅脱氧胆酸/生物合成鹅脱氧胆酸/代谢动力学放射性同位素稀释技术氚成年人人类男(雄)性

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Blood volume

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Seminars in Nuclear Medicine 1975年第1期5卷 63-78页

作者： Wright, Robert R. Tono, Mary Pollycove, Myron Nuclear Medicine Division Clinical Laboratories San Francisco General Hospital San Francisco CA United States Department of Clinical Pathology Laboratory Medicine University of California San Francisco CA United States

During the past decade the diagnostic use of blood volume determinations has declined as a result of the generation of largely inaccurate results and inappropriate normalization and interpretation. After historical development of more than 50 years, current methodology employs ¹²⁵I-labeled human serum albumin and ⁵¹Cr-labeled red blood cells to determine plasma volume and red cell volume, respectively. Accurate blood volume determinations require (1) abandoning the use of the mean body hematocrit: venous hematocrit ratio and using simultaneous independent measurements of both volumes;(2) delaying multiple postinjection patient samples until complete mixing and equilibration are complete;(3) back-extrapolation of plasma concentrations of ¹²⁵I to account for albumin loss from the plasma, and, rarely, back-extrapolation of red cell concentrations to account for dilution by red cells transfused during the procedure;(4) normalization of volumes by adjusting patient weight to normal correspondence with lean tissue mass, whenever necessary. A rapid, routine method that fulfills these four requirements is presented. A number of surgical and medical conditions in which blood volume determinations are very useful in diagnosis and therapy are discussed. Recently developed techniques for blood volume measurements include neutron activation analysis and fluorescent excitation analysis. Correct normalization of accurate blood volume measurements will provide a valuable service to the entire medical community. © 1975 Grune & Stratton, Inc.

关键词：血量测定/方法体质一氧化碳中心静脉压铬放射性同位素误诊红细胞血细胞比容激素类异位醛固酮增多症/诊断高血压/诊断铟铁放射性同位素磷放射性同位素血浆容量红细胞增多症/诊断手术后并发症/诊断钾放射性同位素放射性同位素稀释技术血清白蛋白放射性碘标记休克/诊断锝时间因素转铁蛋白血管升压素类/代谢老年人人类男(雄)性

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Disposition of dietary ethanol carbons in rats: Effects of gender and nutritional status

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METABOLISM-CLINICAL AND EXPERIMENTAL 2000年第3期49卷 379-385页

作者： Cornier, MA Jackman, MR Bessesen, DH Univ Colorado Hlth Sci Ctr Dept Med Div Endocrinol Denver CO 80262 USA Denver Hlth Med Ctr Dept Med Denver CO USA

Dietary ethanol is an important contributor to total caloric intake and has been associated with gender-specific alterations in body weight and the risk for coronary heart disease. To understand the metabolic basis of these effects, it is important to first clarify the effects of gender and nutritional state on the metabolic fate of dietary ethanol. Tracer studies were therefore performed using C-14-labeled ethanol in fasted or fed male and female Sprague-Dawley rats (N = 64) previously unexposed to ethanol, 1-C-14-ethanol (4.5 mu Ci) was mixed with unlabeled ethanol (for a total ethanol dose equal to 10% of total daily caloric intake) and a 3-kcal liquid meal and administered through gastric feeding tubes,(CO2)-C-14 production was measured over the subsequent 8 hours. The C-14 content of skeletal muscle, liver, adipose tissue, gastrointestinal (GI) tract, brain, heart, kidney, and serum was determined at 4 time points following tracer administration (20 minutes and 3, 8, and 24 hours;n = 4 at each time point). Tracer content on a whole-body level was significantly greater in skeletal muscle compared with liver in all groups (1.32 +/- 0.02 x 10(6) v 0.27 +/- 0.02 x 10(6) dpm, P < .001). Skeletal muscle tracer content decreased rapidly after 3 hours, whereas liver tracer content remained fairly constant throughout the study period. Fed female rats were the exception, with a significant increase in the tracer content of total liver and liver lipid at 8 hours. The tracer content was higher in the lipid extracts in liver from fed rats compared with fasted rats (1.08 +/- 0.19 x 10(5) v 0.48 +/- 0.08 x 10(5) dpm, P = .002). While male rats exhibited a fairly constant tracer content in adipose tissue throughout the 24-hour period, female rats showed an increase in adipose tissue tracer content at 8 and 24 hours, with levels 3 to 4 times those of the male animals (5.91 +/- 1.42 x 10(4) v 1.55 +/- 0.42 x 10(4) dpm, P = .02). These results demonstrate that (1) skeletal muscle

关键词：脂肪组织/代谢饮酒二氧化碳/分析碳放射性同位素能量摄取乙醇/代谢禁食肝/代谢肌骨骼/代谢营养状况放射性同位素稀释技术大鼠 Sprague-Dawley 性征组织分布动物女(雌)性男(雄)性大鼠

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Production rates of testosterone in patients with Cushing's syndrome

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METABOLISM-CLINICAL AND EXPERIMENTAL 2000年第2期49卷 229-231页

作者： Vierhapper, H Nowotny, P Waldhäusl, W Univ Vienna Clin Div Endocrinol & Metab Dept Internal Med 3 A-1090 Vienna Austria

Testosterone production rates were determined in 16 patients with Cushing's syndrome (4 men and 12 women) using the stable-isotope dilution technique and mass spectrometry. 1 alpha,2 alpha-D-Testosterone was infused for 10 hours at a dose of 20 mu g/h (men) and 0.4 mu g/h (women) and blood samples were obtained at 20-minute intervals during the last 4 hours of the observation period. Estimated production rates in men with Cushing's syndrome were 27, 73, 150, and 180 mu g/h (mean, 106 +/- 70 mu g/h;healthy men [n = 12], 210 +/- 70 mu g/h). In the 12 women with Cushing's syndrome, testosterone production rates were 0.3 to 22.3 mu g/h (healthy women [n = 5], 4.3 +/- 1.9 mu g/h), There was no difference in testosterone production rates in female patients with central (n = 8) versus adrenal (n = 4) Cushing's syndrome. In summary, testosterone production rates are subnormal or low-normal in male patients with endogenous hypercortisolism, but not in female patients with the same disorder. We conclude that testosterone production in men, but not in women, is predominantly of gonadal origin and hence susceptible to a glucocorticoid-induced suppression of gonadotropin secretion. Copyright (C) 2000 by W.B, Saunders Company.

关键词：库欣综合征/代谢气相色谱-质谱法动力学放射性同位素稀释技术睾酮/生物合成睾酮/药代动力学成年人女(雌)性人类男(雄)性中年人

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Dietary fat saturation, but not the feeding state, modulates rates of cholesterol esterification in normolipidemic men

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METABOLISM-CLINICAL AND EXPERIMENTAL 1999年第10期48卷 1210-1215页

作者： Mazier, MJP Jones, PJH McGill Univ Sch Dietet & Human Nutr St Anne De Bellevue PQ H9X 3V9 Canada St Francis Xavier Univ Dept Human Nutr Antigonish NS B2G 1C0 Canada

To determine whether the rates of cholesterol esterification in normal individuals are affected by diets differing in fats, nine men were randomly assigned to three groups receiving a diet rich in monounsaturated (MONO), polyunsaturated (POLY), or saturated (SAT) fat for 2 weeks using a crossover design, Subjects drank a dose of deuterium oxide, and the fractional esterification rate (FER) was calculated during fed and unfed periods. Total esterified cholesterol was calculated as the product of the FER and pool size, the latter obtained from a decay curve following injection of [4-C-14]-cholesterol. The POLY diet produced the lowest serum cholesterol concentration and the SAT diet the highest (P < .001). For cholesterol ester (CE) deuterium enrichment, an interaction was noted between diet and time (P < .01). The FER was greater (P < .003) in subjects fed the POLY diet versus either of the other diets, although the amount of esterified cholesterol produced, expressed as either milligrams per day (P < .103) or milligrams per kilogram of body weight per day (P < .100), did not differ among groups. No effect of the feeding state was found for either the FER (P < .187) or total esterified cholesterol expressed as milligrams per day (P < .146) or milligrams per kilogram of body weight per day (P < .128). The results suggest that the diet fat type, but not the feeding state, may be responsible for serum esterified cholesterol concentrations. Copyright (C) 1999 by W.B. Saunders Company.

关键词：碳放射性同位素胆固醇/血液胆固醇酯类/血液交叉研究氧化氘膳食脂肪类/药理学进食/生理学脂肪酸类/药理学脂肪酸类单不饱和/药理学脂肪酸类不饱和/药理学模型生物学放射性同位素稀释技术参考值成年人人类男(雄)性

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EFFECTS OF CHANGES IN SERUM OSMOLARITY ON BULK FLOW OF FLUID INTO CEREBRAL-VENTRICLES AND ON BRAIN WATER-CONTENT

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PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY 1975年第3期359卷 253-264页

作者： DIMATTIO, J HOCHWALD, GM MALHAN, C WALD, A NYU MED CTRDEPT NEUROSURG550 1ST AVENEW YORKNY 10016 NYU MED CTRDEPT NEUROL550 1ST AVENEW YORKNY 10016

The effects of changes in serum osmolarity on the rate and osmolarity of bulk flow of fluid into the cerebral ventricles and on cortical white and grey matter water content were studied in cats. Bulk flow rates and osmolarities were measured during ventriculocisternal perfusion both before and after intravenous infusion of glucose solutions. Infusions of glucose in concentrations greater than 6% decreased fluid bulk flow rate and its osmolarity. Glucose in concentrations less than 6 percent increased fluid bulk flow rate and decreased its osmolarity. Bulk flow rate and serum osmolarity were found to be linearly related with a coefficient of osmotic flow of minus 0.835 mul/min per mOsm/l. At the extremes of induced serum osmolarities, (290 and 360 mOsm/l) bulk flow rate was either increased by 120 percent or completely inhibited. Effluent osmolarity also increased proportionately to serum osmolarity (0.338 mOsm/l per mOsm/l). When compared to controls, cortical grey and white matter water content increased by 1.9 percent and 2.9 percent, respectively, when the infused glucose concentration was 2.5 percent or less, and decreased by 1.8 percent and 2.9 percent when the concentration was 10 percent or more. The results of these experiments suggest that the increased bulk flow comes from the brain, rather then directly from the blood.

关键词：血脑屏障脑化学大脑皮质/代谢脑室/生理学脑脊髓液/代谢脑脊髓液/生理学葡萄糖碘放射性同位素容积渗克分子浓度放射性同位素稀释技术水/分析动物猫

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