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Background: In spite of the urgent necessity for a screening test of urinary protein for the early diagnosis of kidney diseases, a rapid, accurate and cost-effective method for their detection has yet to be developed., Methods: A solution containing a buffer agent (pH 2.3) and surfactants and a solution of Erythrosin B are added to a urine sample. After letting the mixture stand for 5 min at 37°C, the dye-bound protein is measured by a spectrophotometer at 546 nm using a Hitachi 7170S automated analyzer., Results: The calibration curve was linear with human serum albumin concentration in the range of 2.4-200 mg/l. The detection limit, 2.4 mg/l was superior to conventional dye-binding methods by one order of magnitude and comparable to the turbidimetric immunoassay (TIA). Spot urine samples from 70 patients who showed (-) or (±) in the dip-stick screening test for proteinuria and 79 healthy volunteers were analyzed. There was an excellent correlation (r=0.978, n=149) between the results given by the proposed method and those by the TIA., Conclusions: This method provides a viable alternative to the conventional immunoassay-based methods for urinary protein measurement, and will be useful in the diagnosis of early stage kidney disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Introduction: Brain natriuretic peptide (BNP) is produced by the ventricles of the heart and is a biomarker for heart failure. Several commercial assays are now available. We evaluated the performance characteristics of the ARCHITECT BNP assay., Methods: We evaluated the limit of blank, limit of detection, linearity and imprecision. Method comparison studies were performed with 3 other automated BNP assays including the ADVIA Centaur, AxSYM, and UniCel DxI 800 methods., Results: The mean LOB and LOD of the Architect assay were 3.5 and 5.8 ng/L, respectively. Imprecision studies yielded within run CVs of 1.1 to 5.1% and total CVs of 2.3 to 5.3% using human plasma based multi-constituent controls at concentrations of 92, 500, and 3500 ng/L. The maximum deviation from the target recovery for dilution linearity was 9.6%. Concordance with other BNP assays at a 100 ng/l cutoff was 91 to 98% and kappa statistics were 0.78 to 0.96. The mean difference between the Architect and Advia Centaur methods was positive. For the other methods, the mean difference with the Architect was negative., Conclusions: The Architect BNP assay shows good performance characteristics with total imprecision < or =5.3%. It agrees well with the Advia Centaur, AxSYM, and UniCel DxI BNP assays.

Background: Low levels of serum testosterone, as typically found in women and children, cannot be measured reliably by immunoassays. Our aim was to develop a sensitive assay to quantitate low serum testosterone concentrations using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were compared to those obtained with various immunoassay techniques., Methods: Serum testosterone levels in 70 women and children were measured using LC-MS/MS and compared with two automated, non-isotopic immunoassays, and three manual, isotopic immunoassays. Serum extraction was required only for LC-MS/MS and one of the isotopic methods., Results: Deming regression analysis was used for comparison: the correlation coefficients were between 0.772 and 0.870, and the slopes between 0.972 and 1.365. Using Bland and Altman analysis, all the 5 immunoassays showed a positive mean difference compared with LC-MS/MS: all overestimated the testosterone levels in women and children., Conclusion: None of the immunoassays tested proved sufficiently reliable when low testosterone concentrations (< or =3.47 nmol/L) were measured. In contrast to conventional isotopic and non-isotopic immunoassay techniques, LC-MS/MS allows the precise determination of low testosterone levels. It has adequate sensitivity and is not subject to interference from other steroids that were tested.

Background: In contrast to RIA, recently available ELISAs provide the potential for fully automated analysis of adiponectin. To date, studies reporting on the diagnostic characteristics of ELISAs and investigating on the relationship between ELISA- and RIA-based methods are rare., Methods: Thus, we established and evaluated a fully automated platform (BEP 2000; Dade-Behring, Switzerland) for determination of adiponectin levels in serum by two different ELISA methods (competitive human adiponectin ELISA; high sensitivity human adiponectin sandwich ELISA; both Biovendor, Czech Republic). Further, as a reference method, we also employed a human adiponectin RIA (Linco Research, USA). Samples from 150 patients routinely presenting to our cardiology unit were tested., Results: ELISA measurements could be accomplished in less than 3 h, measurement of RIA had a duration of 24 h. The ELISAs were evaluated for precision, analytical sensitivity and specificity, linearity on dilution and spiking recovery. In the investigated patients, type 2 diabetes, higher age and male gender were significantly associated with lower serum adiponectin concentrations. Correlations between the ELISA methods and the RIA were strong (competitive ELISA, r=0.82; sandwich ELISA, r=0.92; both p<0.001). However, Deming regression and Bland-Altman analysis indicated lack of agreement of the 3 methods preventing direct comparison of results. The equations of the regression lines are: Competitive ELISA=1.48 x RIA-0.88; High sensitivity sandwich ELISA=0.77 x RIA+1.01., Conclusions: Fully automated measurement of adiponectin by ELISA is feasible and substantially more rapid than RIA. The investigated ELISA test systems seem to exhibit analytical characteristics allowing for clinical application. In addition, there is a strong correlation between the ELISA methods and RIA. These findings might promote a more widespread use of adiponectin measurements in clinical research.

Background: Adiponectin is an adipose-derived hormone that plays a role in regulating metabolic processes such as fat partitioning and lipid and glucose metabolism. Quantification of adiponectin is useful for obtaining information on metabolic syndrome, but there is no rapid method to measure adiponectin for clinical use., Methods: We developed a rapid and sensitive latex particle-enhanced turbidimetric immunoassay (LTIA) using a latex bead-immobilized anti-adiponectin polyclonal antibody. The assay was performed on a Hitachi H7170 analyzer and evaluated for validity as a method to quantitate adiponectin, in parallel with the ELISA., Results: Dilution tests using LTIA showed linearity from 0.25 to 30 microg/ml. Within-run CV and total CV were obtained in the range of 0.8-1.9% and 1.1-2.0%, respectively. No interference was observed in the testing of specimens containing potentially interfering substances such as bilirubin, ditaurobilirubin, hemoglobin triglyceride, rheumatoid factor, type IV collagen, fibronectin, and complement factor (C1q). A strong correlation between LTIA and ELISA was confirmed (n=30, r=0.990, y=0.95x+0.39)., Conclusion: The LTIA assay is applicable to quantitating the serum concentration of adiponectin. This assay is more convenient and faster than ELISA and suitable for clinical routine analysis.

Background: Erythropoietin (EPO) is a glycoprotein hormone produced by specialized cells in the kidney that acts as the primary regulator of erythropoiesis. Serum EPO measurements are useful for diagnostic evaluations of anemia and polycythemia., Methods: The IMMULITE 2000 is an automated random access immunoassay analyzer for the central laboratory. The limit of detection (LOD), linearity, imprecision, comparison to another commercially available assay, and reference interval of a new EPO assay for this analyzer were assessed., Results: The LOD was 0.2 U/l. The assay was linear within an allowable systematic error of 10% over the range tested (2-178 U/l). The total imprecision of the new assay was <7% for concentrations from 15.8 to 68.4 U/l. Comparison with the Advantage EPO assay method showed comparable results. Deming regression analysis gave a slope of 1.13+/-0.02, an intercept of -1.09+/-0.97, and a S(y/x) of 7.1 (r=0.99) over the range tested (2-200 U/l)., Conclusions: The IMMULITE 2000 EPO assay shows acceptable performance and is suitable for routine clinical use.

Background: C-reactive protein (CRP) is a nonspecific marker of inflammation that can be used as a marker for atherosclerotic risk. This application requires increased precision at low CRP concentrations compared to traditional assays., Methods: The Dimension RXL is an automated chemistry analyzer for central laboratory use. The limit of detection, limit of quantification, linearity and imprecision of a high-sensitivity CRP assay developed for it were assessed. Method comparison studies were performed using samples both inside and outside the reference interval. The presence of a prozone effect was also evaluated., Results: The limit of detection was 0.7 mg/l. The method was linear from 2 to 60 mg/l and from 1 to 60 mg/l using systematic error limits of 10% and 20%, respectively. The total imprecision was <10% for CRP concentrations above 1.5 mg/l. No prozone effect was seen at a CRP concentration of 450 mg/l, the highest concentration tested. Using samples from 212 apparently healthy adults, the Dimension RXL method demonstrated good concordance with the BN II high-sensitivity CRP method for samples in the highest quartile. It also compared well using samples with elevated CRP concentrations., Conclusions: The Dimension RXL high-sensitivity CRP method may be adequate for atherosclerotic risk prediction in clinical practice if accurate and precise measurement is only required for the highest quartile. However, the total error of this method for CRP concentrations <3 mg/l appears too large for accurate assignment to lower risk groups.

Background: C-reactive protein (CRP) is a non-specific marker of inflammation that can be used for atherosclerotic risk assessment. This application requires increased precision at low CRP concentrations compared to traditional assays., Methods: The Micros CRP analyzer (ABX Diagnostics) is a small bench top device. Its limit of detection, limit of quantitation, linearity and imprecision were assessed. Method comparison studies were performed using samples both inside and outside the reference interval. Anticoagulant effects and the prozone effect were also evaluated., Results: The limit of detection was 0.1 mg/l. The method was linear from 2 to 60 and 0.3 to 60 mg/l using systematic error limits of 10% and 20%, respectively. The total imprecision was <8% for CRP concentrations from 0.7 to 9.1 mg/l. A prozone effect was seen at CRP concentrations >500 mg/l. Using samples from 120 apparently healthy adults, the Micros CRP method demonstrated excellent concordance with the BN II high sensitivity CRP (hs-CRP) method. The Micros CRP method compared well with a nephelometric method using samples with elevated CRP concentrations., Conclusions: The Micros CRP method is adequate for atherosclerotic risk prediction in clinical practice but does not have adequate accuracy at CRP concentrations <2 mg/l for epidemiological studies.

Creatinine deiminase (EC 3.5.4.21) from the anaerobic microorganism BN11 has been purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephacryl-S-300 superfine and chromatography on DEAE-Sepharose C1 6B. The final enzyme preparation had a specific activity of 78 units per mg protein. Analysis of creatinine deiminase by polyacrylamide gradient gel electrophoresis and fast-flow-liquid-chromatography gave a relative molecular mass of 285 kDa and 288 kDa, respectively. By treatment with sodium dodecylsulfate and 2-mercaptoethanol creatinine deiminase was dissociated yielding one polypeptide with a relative molecular mass of 47.5 kDa. The enzyme was entirely specific for creatinine and showed a Km value of 0.15 mM. Creatinine deiminase was used to determine the concentration of creatinine in serum and urine using a manual method and an automated system.

Isothermal and isocratic HPLC chromatography for rapid quantitation of isoleucine and leucine (merged peak), tyrosine and phenylalanine from a single dried blood filter paper specimen (1/8" dot blot) is described. The net chromatography time of the relevant peaks is less than 3 min. The results can be used to screen for PKU, MSUD, and tyrosinemia in one single assay. The advantage for early determination of PKU by simultaneous quantitation of both phenylalanine and tyrosine with the determination of their ratio is discussed. Using a dual column system this procedure might be suitable for large-scale newborn screening.

A continuous-flow system is described that utilizes an immobilized glycerol dehydrogenase nylon-tube reactor integrated into the flow system of a Technicon AutoAnalyzer II for the routine estimation of glycerol in serum and various beverages. Results are compared with determinations made by the solution method that uses the three enzymes glycerokinase, pyruvate kinase and lactate dehydrogenase. These routine trials give reliable and reproducible results with high precision and economy. The glycerol dehydrogenase reactor is stable during intermittent or continuous use for a least 3500 tests. Stored at 4 degrees C when not in use, the reactor is quite stable for several months.

This paper describes a method to determine transketolase activity in erythrocytes, with or without addition of thiamine-pyrophosphate, by quantitating the formation of glucose-6-phosphate forming during the incubation of hemolysed blood with ribose-5-phosphate. Glucose-6-phosphate is determined enzymatically, after deproteinisation of the sample, using a centrifugal fast analyser. The proposed method gave a correlation of r = 0.89 with an accepted colorimetric method. Reference values for blood are in the range 32--101 U/l.

An automated method for the assay of N-acetyl-beta-glucosaminidase in human urine is described and a normal range of urinary N-acetyl-beta-glucosaminidase activity is reported. The automated method has been used routinely over a twelve month period in two London hospitals for monitoring the excretion of N-acetyl-beta-glucosaminidase in renal transplant patients. The abnormal elevation of urinary N-acetyl-beta-glucosaminidase provides an early warning of rejection by indicating the presence of renal parenchymal damage. The automated assay of urinary N-acetyl-beta-glucosaminidase may also be used in screening for the presence of renal disease.

We have modified the direct barbituric acid (DBA) test for urine nicotine metabolites so that it can be used on a continuous flow auto-analyser. The performance of the method was compared with a cotinine radio-immunoassay (RIA). The auto-analyser DBA method and cotinine RIA performed equally well in terms of assessing smoking status, yielding a false positive rate of 1.5% at a detection rate of 98% when compared to self-reported smoking information. The results obtained with the two methods were correlated, r = 0.91, although the DBA method results were consistently higher than the corresponding cotinine RIA results; the method detects cotinine and other nicotine metabolites. The coefficient of variation for the DBA method was 3.4% compared with 10% for the RIA. The DBA method has a throughput of about 250 samples per day compared to about 70 per day by RIA and the reagents are readily obtained and inexpensive.

Enzymatic methods adapted to the KAPA system can be carried out in a final test volume of less than 10 mu 1. The procedure is mechanized, including the pre-analytical steps. Day-to-day precision is 2-6% C.V. for single determinations. Advantages of the technique are: (1) capillary plasma can be used; (2) reagent waste is reduced by about two orders of magnitude compared to common procedures, and (3) 400 specimens can be assayed by one technician per day. These conditions facilitate serial determinations, large scale screening, or repetitive monitoring of values in a group of individuals.

A simple semi-automated procedure for assay of catecholamines in plasma and urine is described. Each step is examined and the end product is the culmination of these investigations. Use is made of batch alumina adsorption and elution, after comparison with column methods had shown no real advantage. Urine estimations included a preliminary ion-exchange column step. The estimation is such that 40 to 50 specimens of plasma or 60 to 70 specimens of urine can be assayed in a day by a trained laboratory technician. The method is both specific and reproducible, and the sensitivity is increased over previously reported methods. Interference by other catecholamines and compounds is examined and discussed, as are alternative stabilising agents and reactions for the rearrangement and stabilisation of the fluorescent compounds in strong alkali.

A new method for the determination of ionized calcium in parotid saliva utilizing an automatic ionized calcium analyzer (Orion Biomedical Model Space-Stat 20) was evaluated. The method showed good precision with a relative standard deviation of 2.9--3.8 per cent in the range 0.20--0.79 mmol . l-1 after 226 duplicate measurements. Mean recovery was 94.9% in the range of 0.6--1.8 mmol. l-1. Bovine mucin was found not to interfere with the calcium electrode. Differences in sodium concentration, ionic strength and pH between the standard solutions and samples caused systematic errors. These were corrected using experimentally derived formulas. The influence of pH on the concentration of ionized calcium was studied in 13 parotid saliva samples. A non-linear relationship ranging between 32 and 84% of the total calcium concentration was found.

The special features of a laboratory computer system operated by laboratory staff around the clock are presented. The system provides registration of work, production of worksheets, on-line data acquisition and reporting results. The reasons for the special design features are discussed.

A new, enzymic method of triglyceride determination in serum and plasma by use of an immobilized glycerol dehydrogenase nylon-tube reactor, integrated into the flow system of an AutoAnalyzer II (Technicon) is described. The combination of this reactor, stable for 1500--2000 tests, with the lipolytic enzymes which are added in solution yields a reliable and reproducible assay, which correlates well with the commonly used fully enzymic triglyceride determination. Using this new method, the cost can be reduced to about one-third of that of the other method.

We describe a one-step kinetic method for the determination of 5'-nucleotidase (EC 3.1.3.5). Inosine is formed by the hydrolysis of inosine 5'-monophosphate which is catalyzed by seric 5'-nucleotidase, and then is converted to hypoxanthine by nucleoside phosphorylase. Two moles of hydrogen peroxide are formed for each mole of hypoxanthine oxidized to urate by xanthine oxidase. The rate formation of hydrogen peroxide is monitored at 510 nm using the oxidation of the chromogenic system 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone in the presence of peroxidase. beta-Glycerophosphate inhibits the unspecific cleavage of the substrate by alkaline phosphatases. Inorganic phosphate is added to improve the reagent stability, and ferrocyanide to reduce bilirubin interference. Automation of the technique requiring 20 microliter of serum on a centrifugal analyzer is also described.

A simple enzymatic spectrophotometric micromethod is described for direct kinetic assay of acetate in serum or plasma using the Eni-Gemsaec centrifugal fast analyser. The method is based on the transformation of acetate and ATP into acetylphosphate and ADP by acetate kinase (EC 2.7.2.1). ADP is further measured by two coupling reactions involving pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) with measurement of NADH consumption at 340 nm. The method involves a reagent blank for compensation of reagent deterioration, a preincubation of 3 min without acetate kinase to eliminate any interference due to endogenous pyruvate, and a two-point kinetic protocol with measurements of absorbance at 95s and 395 s. The analytical performances of the proposed method were investigated using an evaluation scheme proposed by the French Society of Clinical Biology.

Concentrations of polyvinylpyrrolidone and o-phenylenediamine have been optimized to allow performance of serum inorganic phosphorus using a discrete analyzer, the Vickers M-300. The reagents are stable. The method is precise, accurate, reliable and very sensitive. A precipitate forms in the reaction mixture from some serum specimens containing abnormal globulins.

An assessment of the Gilford Automatic Enzyme Analyser was conducted over a period of one year. The optics of the instrument were satisfactory with regard to accuracy of wavelength selection and linearity of absorbance response. Excellent precision was obtained for both absorbance readings and operation of the dispenser pump. Carry-over within the microflow-cell was low. The method of operation recommended by the manufacturers for enzyme determinations failed to take account of endogenous blank reactions which could lead to significant error. When revised methods utilising a pre-incubation stage and initiation with a single substrate were employed, the results correlated well with those obtained with standard automatic (LKB 8600) and manual (Pye Unicam SP 800) kinetic systems for aspartate and alanine aminotransferase, creatine phosphokinase and alpha-hydroxybutyrate dehydrogenase, and the precision at all activity levels was satisfactory. Acceptable precision could not be obtained over the clinical range for enzyme assays requiring a blank determination on each sample (5'-nucleotidase and adenosine deaminase) and those with very low normal serum activities (isocitrate dehydrogenase and glutamate dehydrogenase). These limitations appeared to be due to relative insensitivity of the transducer response and liability to optical disturbance. This apart, the instrument has many advantages over alternative equipment.

An immunoradiometric two-site assay for thyrotrophin (TSH) in dried blood filter paper spots is described. The assay is automated by means of the Kemtek 3000 automated immunoassay system. The technique uses a 6.0 mm disc punched from the dried blood samples collected as part of the screening programme for phenylketonuria. The method is sensitive and precise, and results correlate well with those obtained in TSH assays of serum samples. The procedure is rapid, results being available within 24 h of receipt of samples. Of 25 204 specimens so far screened by this assay, 99.9% have TSH levels less than 15 mU/l. One false positive result has been obtained and six confirmed cases of neonatal hypothyroidism detected, giving a prevalence of 1 in 4200.

A method has been designed for the assay of pancreatic carboxypeptidase A in blood serum. It uses Z-Gly-Phe as the substrate and fluorimetric determination of the released phenylalanine in an amino acid analyser, which yields a measure of free carboxypeptidase A. In addition, the sum (free carboxypeptidase A + procarboxypeptidase A) can be determined on a second portion preincubated with trypsin, which converts the proenzyme to the active form. Determinations made in fifteen healthy individuals showed the presence of a measurable concentration of free carboxypeptidase A. In acute pancreatitis, total carboxypeptidase A is raised. An increase in circulating proenzyme is observed in some cases. Data from 46 patients show a good correlation between total carboxypeptidase A, lipase and immunoreactive trypsin. Differential determination of procarboxypeptidase A and free carboxypeptidase A provides an interesting new tool for the diagnosis of pancreatic disorders.

A semi-automated competitive, double-antibody, solid-phase enzyme-linked immunosorbent assay for apolipoprotein B (Apo B) has been developed which utilizes microtiter plates with commercially available monoclonal antibodies and alkaline phosphatase-conjugated second antibody. The working range of the assay is 20-200 ng. The concentration of plasma Apo B was 0.88 +/- 0.20 g/l (n = 40) for a random sample of normal adults. The correlation coefficient for this assay, compared to a radial immunodiffusion assay, was 0.95 (slope = 1.13, intercept = -15). The quantification of the samples was not influenced by freezing and thawing, storage at -20 degrees C for up to 9 mth, or the lipoprotein particle on which the Apo B was present. The method is suitable for measurement of apolipoprotein B in either normal or pathological plasma, lipoprotein density classes, and is sensitive enough to quantify Apo B in cell biological and molecular biological investigations.

An automated method is presented for the determination of thrombin generation in plasma during activation by thromboplastin. The thrombin generated is allowed to split the chromogenic substrate Tol-Gly-Pro-Arg-pNA yielding p-nitroaniline. The increase in absorbance at 410 nm is recorded. This assay may serve as a specific, precise and fast alternative for the conventional clotting tests: "Prothrombin time" and "Thrombotest".

We describe an adaptation of a microcomputer-controlled stopped-flow analyzer for automated analysis of albumin in serum. The immediate reaction between bromcresol green or purple and albumin is used to develop a routine procedure with 12 s reaction time. The approach shows excellent linearity to 68 g/l, good sensitivity, a relative SD of less than 0.5%, mean recovery 99.1%, high sample throughput (180 prediluted samples/h) and no significant interferences. The 'reaction time' related overestimation of albumin that appears in prolonged procedures is eliminated, and results with this method correlate well with those obtained using the more specific immunonephelometric method.

Measurement of plasma angiotensin-converting enzyme (ACE) is of value in sarcoidosis and, when specific inhibitors are prescribed therapeutically, in hypertension and heart failure. In this study a rapid kinetic assay for estimation of plasma ACE is described and assessed. In particular the effects of zinc ion have been studied. In contrast to previous reports, a marked potentiation of ACE activity by micromolar concentrations of zinc was noted. The relationship between plasma zinc concentration and ACE activity in vivo was also investigated and related to albumin concentration. The findings may have implications for interpretation of plasma ACE activity in patients with low plasma zinc levels.

Rates of reaction of different oestrogens in Kober reagent vary greatly. Rate constants were measured between 100 degrees C and 150 degrees C. Oestradiol, oestrone, 16-oxo-oestradiol, 16 alpha hydroxyoestrone, 16-epioestriol and urine pool show two sequential first order reactions at 100 degrees C; oestriol and its conjugates give a single reaction (slower than the other oestrogens except for the very slow oestretrol). Above 120 degrees C differences decrease, all oestrogens having one rate for Kober product formation: the decay reaction, which is also first order, becomes significant. Oestriol and its conjugates have relatively high apparent activation energies in the Kober reaction (120-138 kJmol-1) compared to other oestrogens studied (105-124 kJmol-1). The apparent activation energy for the decay reaction is the same within experimental error (115 +/- 3 kJmol-1). This is consistent with a common product formed from oestrogen reacting with Kober reagent. Analytical methods must respond similarly to major urinary oestrogens. Appropriate conditions include 100 degrees C for at least 20 min or 135 degrees C for 3 to 4 minutes.

A continuous flow automated method for urine albumin was developed based on the ability of albumin to displace bromophenol blue from a bromophenol blue detergent complex. The method is almost specific for albumin, giving a slight response with an alpha-globulin fraction from serum. Results agreed closely with rocket electrophoresis albumin estimates on urine samples, but significantly less closely with "total urine protein" estimates by an acid protein precipitation, followed by biuret colour reaction, procedure. This method is being used to replace the indefinite "total protein" test for most routine purposes.

A rapid automated method for the determination of serum methionine is presented for use with the Technicon AutoAnalyzer system. It is based on the decolorisation of halide platinates by organic sulphides, including methionine. The 5--95% reference range from 249 sera from hospital patients (excluding those with liver and renal diseases and diabetes) was 20--60 mumol/l, mean 35 mumol/l. The S.D. of the method for 40 duplicate samples was 2.2 mumol/l. Recoveries ranged from 101--105%. This measurement facilitates the clinical evaluation of liver disease, especially acute liver failure, kidney disease and diabetes mellitus.

A rapid and convenient automated procedure for the determination of alpha-tocopherol is described. As little as 0.3 ml of serum or plasma can be used at a sample rate of 20 per hour and carry-over is negligible. Within-batch precision was 4.71% at 5.0 mg alpha-tocopherol per 1, and 3.68% at 20 mg/l, compared with a between-batch precision of 6.98% at 10 mg/l. Using this procedure alpha-tocopherol has been found to be distributed in a log-normal manner and on that basis the range of values for 80 haematologically normal subjects was calculated to be 4.0 to 16.0 mg/l. In pregnancy the alpha-tocopherol was elevated, ranging from 6.7 to 24.0 mg/l. During the first year of life normal infants ranged from 2.0 to 16.0 mg/l which was higher than a small group with various disorders whose values ranged from 1.2 to 8.3 mg/l. The cord blood level was approximately one-fifth of the maternal level.

Detergent must be added to the strongly acidic Kober reagent to obtain reproducible and well separated peaks in continuous flow analytical methods for oestriol in pregnancy urine. Brij 35 and other polyether detergents are not satisfactory because they or their impurities interfere in the reaction causing high blank response in colorimetric reactions and diminished fluorescence responses. In a single phase fluorometric method these detergents also caused turbidity or phase separation in the solution which passed through the fluorometer. Some anionic sulphate and sulphonate detergents were also unsatisfactory being immiscible with Kober reagent. Selected cationic and betaine detergents are shown to be possible alternatives, although one which is satisfactory in the kober reagent may be unsuitable for a dilution reagent. The betaine Empigen BB was found to be suitable for inclusion in both of these reagents.

A method is described for the quantitative assay of pipecolic acid in biological fluids using column chromatography, with acid ninhydrin for color development. The technique permits measurement of plasma pipecolic acid in normal individuals with blood samples of 1 ml. The mean value in 29 pediatric patients, aged 4 months to 17 years, hospitalized with a variety of diagnoses was 2.1 mumol/1 +/- 1.6. Newborn infants had slightly elevated plasma levels with a mean of 12 mumol/1 +/- 5.6. Among the diagnostic categories, patients with liver disease were distinctive in commonly having levels at the upper limits of normal. In some cases, the levels were exceedingly high, reaching 78 mumol/1 in one fatal case.

13 Urinary steroid metabolites have been determined by automated gas-liquid chromatography with a 20-m glass capillary column and a computing integrator. Concentrations up to 2 mg/24 h computed by the integrator compare well with those obtained by peak height measurements. At higher concentrations discrepancies occurred, paticularly for the C21 steroids where falsely low values were calculated using peak heights. Mean excretion by healthy males and females of seven steroid metabolities is presented.

A unique variant of human albumin was discovered in a family following routine multiple biochemical analysis. Their sera demonstrated increased Bromcresol Green (BCG) dye binding under certain reaction conditions (excess amounts of the detergent Pegosperse in the presence of Merthiolate). This effect was also seen with a structurally similar dye, Bromcresol Purple (BCP). The variant sera also showed an altered pattern of BCG dye binding (in the presence of Merthiolate and excess Pegosperse) compared to normal sera when the temperature or the pH of the dye reagent was changed. Decreased binding of the dye 2-(4'-hydroxybenzeneazo)benzoic acid (HABA) was also noted. Similar findings were observed in six other patients over a period of 21 months. Column chromatography of the variant sera on a DEAE-Sepharose column separated the albumin into two fractions, one showing normal dye binding properties, the other showing the more extreme pattern of dye binding seen for the whole variant serum.