Reference intervals for bone turnover markers in Spanish premenopausal women.

BACKGROUND: The aims of this study were to establish robust reference intervals and to investigate the factors influencing bone turnover markers (BTMs) in healthy premenopausal Spanish women. METHODS: A total of 184 women (35-45 years) from 13 centers in Catalonia were analyzed. Blood and second void urine samples were collected between 8 a.m. and 10 a.m. after an overnight fast. Serum procollagen type I amino-terminal propeptide (PINP) and serum cross-linked C-terminal telopeptide of type I collagen (CTX-I) were measured by two automated assays (Roche and IDS), bone alkaline phosphatase (bone ALP) by ELISA, osteocalcin (OC) by IRMA and urinary NTX-I by ELISA. PTH and 25-hydroxyvitamin D (25OHD) levels were measured. All participants completed a questionnaire on lifestyle factors. RESULTS: Reference intervals were: PINP: 22.7-63.1 and 21.8-65.5 µg/L, bone ALP: 6.0-13.6 µg/L, OC: 8.0-23.0 µg/L, CTX-I: 137-484 and 109-544 ng/L and NTX-I: 19.6-68.9 nM/mM. Oral contraceptive pills (OCPs) influenced PINP (p=0.007), and low body mass index (BMI) was associated with higher BTMs except for bone ALP. Women under 40 had higher median values of most BTMs. CTX-I was influenced by calcium intake (p=0.010) and PTH (p=0.007). 25OHD levels did not influence BTMs. Concordance between the two automated assays for PINP and particularly CTX-I was poor. CONCLUSIONS: Robust reference intervals for BTMs in a Southern European country are provided. The effects of OCPs and BMI on their levels are significant, whilst serum 25OHD levels did not influence BTMs. Age, calcium intake, BMI and PTH influenced CTX-I. The two automated assays for measuring PINP and CTX-I are not interchangeable.

Alkaline phosphatase in healthy children: reference intervals and prevalence of elevated levels.

BACKGROUND: Transient hyperphosphatasemia (TH) is an often unnoticed benign entity, primarily affecting children below 5 years of age. However, the prevalence among healthy children is unknown. We used data from a Swedish pediatric reference interval project to estimate the prevalence of high alkaline phosphatase (ALP) among healthy children and to calculate pediatric reference intervals. METHODS: Blood was collected from 699 subjectively healthy children aged 6 months to 18 years. After exclusion of subjects with high ALP, age- and gender-specific reference intervals were calculated. RESULTS: Six children had ALP levels >16.7 µkat/l (>1,000 U/l), including 4 females and 2 males aged 7-22 months. The prevalence in the age group from 6 months to 2 years was 6.2% (6/97). None of the older children had levels of ALP >16.7 µkat/l. The study did not include the follow-up of these apparently healthy children. Consequently, conditions others than TH explaining the elevated ALP could not be excluded. However, general chemistry analyses, such as liver enzymes, calcium, intact PTH and vitamin D, were essentially normal in these children. CONCLUSIONS: The prevalence of high ALP among subjectively healthy children was approximately 2.4% below 5 years of age and 6.2% below 2 years. Reference intervals vary with age and gender.

Titanium dioxide nanotube films: Preparation, characterization and electrochemical biosensitivity towards alkaline phosphatase.

Titania nanotubes (TNTs) were prepared by anodization on different substrates (titanium, Ti6Al4V and Ti6Al7Nb alloys) in ethylene glycol and glycerol. The influence of the applied potential and processing time on the nanotube diameter and length is analyzed. The as-formed nanotube layers are amorphous but they become crystalline when subjected to subsequent thermal treatment in air at 550°C; TNT layers grown on titanium and Ti6Al4V alloy substrates consist of anatase and rutile, while those grown on Ti6Al7Nb alloy consist only of anatase. The nanotube layers grown on Ti6Al7Nb alloy are less homogeneous, with supplementary islands of smaller diameter nanotubes, spread across the surface. Better adhesion and proliferation of osteoblasts was found for the nanotubes grown on all three substrates by comparison to an unprocessed titanium plate. The sensitivity towards bovine alkaline phosphatase was investigated mainly by electrochemical impedance spectroscopy in relation to the crystallinity, the diameter and the nature of the anodization electrolyte of the TNT/Ti samples. The measuring capacity of the annealed nanotubes of 50nm diameter grown in glycerol was demonstrated and the corresponding calibration curve was built for the concentration range of 0.005-0.1mg/mL.

Harmonization of values for serum alkaline phosphatase catalytic activity concentration employing commutable calibration materials.

BACKGROUND: Harmonization of results allows a more effective utilization of laboratory tests; we verified the feasibility of harmonizing serum alkaline phosphatase results by two methods. METHODS: Patient sera (n=106) and candidate calibration materials (n=8) were analyzed by two methods, employing either diethanolamine (DEA) or 2-amino-2-methyl-1-propanol (AMP) as phosphate-accepting buffers. Results for patient sera by the DEA method were recalculated, with either a commutable or a non-commutable calibration material, both with values assigned by the AMP method. RESULTS: After calibration with the commutable material, the median intermethod difference (DEA-AMP) and ratio (DEA/AMP) dropped from 195 U/l to 0 U/l and from 2.47 to 1.00, respectively. When a non-commutable material was used the former became 124 U/l and the latter 1.94. After recalibration with the commutable material, linear regression and correlation analysis of DEA vs AMP values for the set of 106 patient sera gave: intercept=0.8 U/l; slope=0.997; and nonparametric correlation coefficient r=0.9995. CONCLUSIONS: Harmonization of alkaline phosphatase results by AMP and DEA methods is feasible when commutable calibration materials are used in the trueness transfer process.

Enzyme immunoassays for measuring complement-dependent prevention of immune precipitation (PIP) and solubilization of preformed antigen-antibody complexes (SOL).

We have developed simple and sensitive enzyme-based methods for evaluating the ability of serum complement to prevent immune complex precipitation (PIP) or to solubilize preformed immune complexes (SOL). Alkaline phosphatase, serving both as antigen and label, is added to goat IgG anti-alkaline phosphatase antibodies, with serum present throughout the assay (PIP), or added after immune complex formation (SOL). After incubation at 37 degrees C for 1 h followed by centrifugation, the enzyme activity of the supernatant, reflecting the amount of immune complexes in solution, is measured by colorimetry. Results are expressed with reference to a standard serum pool assigned 100 arbitrary units (AU). Intra- and inter-assay variabilities are within 10%. The normal ranges were 67-133 AU for PIP and 72-129 AU for SOL. These methods have been standardized for clinical use in relation to impaired complement function and immune complex disease, and adapted for measuring complement mediated binding of immune complexes to erythrocytes. They are sensitive, easy to perform and do not require expensive facilities. By measuring the interaction of complement with immune complexes, these methods may highlight aspects of the classical and the alternative pathway that are different from those detected using haemolysis as an endpoint.

Highly sensitive amperometric enzyme immunoassay for alpha-fetoprotein in human serum.

A sandwich amperometric enzyme immunoassay with flow injection for alpha-fetoprotein in human serum has been developed with alkaline phosphatase as the enzyme label. p-Hydroxyphenyl phosphate was used as the substrate for alkaline phosphatase. The hydrolysis product, hydroquinone, was detected by oxidative amperometry in a flow injection system. The amperometric wall jet detector was fitted with a glassy carbon working electrode held at 350 mV vs. Ag/AgCl. The detection limit of hydroquinone in 30 mM borate buffer pH 9.5 was 1.2 x 10(-10) M (linearity range: 10(-9)-5.12 x 10(-6 M). A detection limit for free alkaline phosphatase of 1.2 x 10(-15) M (linearity range: 10(-15)-10(-13) M), or about 36 000 molecules, was observed (same borate buffer and incubations of 10 min at 25 degrees C). These conditions were maintained for the amperometric alpha-fetoprotein immunoassay. For comparison purposes, a photometric detection system was set up, with p-nitrophenyl phosphate as enzyme substrate and the same pair of antibodies and incubation conditions. The detection limit for alpha-fetoprotein obtained by amperometry, 0.07 ng/ml (linearity range = 5-500 ng/ml), was 14 times lower than by photometry. The amperometric enzyme immunoassay correlates well with a commercial colorimetric immunoassay (r = 0.986, slope = 0.967, n = 240).

Effect of type of container, storage temperature and humidity on the biological activity of freeze-dried alkaline phosphatase.

The suitability of butyl rubber-stoppered vials was compared with that of ampoules for the storage of freeze-dried biological materials under a variety of temperature and humidity conditions using alkaline phosphatase as a model. Immediately after freeze-drying, alkaline phosphatase activity in vials and ampoules was similar but decreased at temperatures of 37 degrees C, 45 degrees C and at cycling temperatures with high relative humidities over the 12-month test period, with the least activity in vials with untreated stoppers. By the end of the study at 12 months post-preparation, moisture levels were lowest (0.4%) in the vials where stoppers had been treated with industrial methylated spirit and air-dried, intermediate (1.0%) in DIN ampoules and highest and most variable (1-4%) in vials with untreated stoppers. There was no apparent exchange between the external atmosphere and that within the vials via the stoppers and the moisture in the vials with untreated stoppers appeared to have originated from the stoppers themselves. It was concluded that the use of vials with treated stoppers was almost as effective as the use of DIN ampoules for the freeze-drying and storage of alkaline phosphatase activity under a range of temperatures and humidities. Fluctuating temperatures were more detrimental to alkaline phosphatase activity than continuous storage at a single high temperature.

A modified alkaline phosphatase enzyme amplification system and its application in an HIV antigen ELISA.

Alkaline phosphatase amplification systems increase the sensitivity of enzyme-linked immunoassays (ELISAs). Here we describe a modified method by which levamisole is a component of the substrate buffer. This increases sensitivity by reducing the signal from non-specific alkaline phosphatases. The modified procedure was applied to an 'in house' HIV-1 p24 antigen capture assay and was shown to increase the sensitivity 4-fold as compared to the unmodified system. The performance of the modified amplification system is comparable to that of commercially available systems.

Standard calibration proteins for Western blotting obtained by genetically prepared protein A conjugates.

Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest.

Certification of an enzyme reference material for alkaline phosphatase (CRM 371).

We have produced a batch of lyophilized alkaline phosphatase (AP) for use as an enzyme reference material. The enzyme was partly purified from pig kidney to a specific activity of 400 U/mg of protein and is essentially free from contaminating enzyme activities. The kinetic properties of the preparation are very close to those of the enzyme present in human serum. The partly purified AP was lyophilized in a matrix containing bovine serum albumin (40 g/L), MgCl2, ZnCl2 and NaCl. The vial-to-vial variability with respect to the catalytic concentration of the final product was 0.008. The predicted annual relative loss of activity was less than 0.01% at -20 degrees C and 0.04% at 4 degrees C. This material was certified using the IFCC proposed method. The certification procedure involved 19 laboratories throughout the world. The certified alkaline phosphatase catalytic concentration in the reconstituted material was 254 U/L with a 0.95 confidence interval of +/- 6 U/L.

Alkaline phosphatase and the International Clinical Enzyme Scale.

The wide numeric variation of alkaline phosphatase (ALP: EC 3.1.3.1) values reported from clinical laboratories is clearly revealed by the grossly incompatible data found in large interlaboratory surveys. The authors suggest that this unsatisfactory situation is readily correctable by simply expressing all numeric results on a single scale, the International Clinical Enzyme Scale (ICES). ICES for ALP (ALP/ICES) rests upon a well-defined reference system that relates the IFCC Reference Method for ALP to numerous stable primary and secondary ALP reference materials. The authors show by calibrations with ALP/ICES reference materials that raw coefficients of variation of 25-30% due to reagent, temperature, or instrument differences could be reduced to as low as 2%. ICES and the reference system approach automatically unifies the numeric outputs of the working clinical laboratories by relating all measurements and reference materials to one clearly defined international reference method, yet this concept allows many technologic options in the individual laboratory.

Development of a reference material for alkaline phosphatase.

In developing a Reference Material for alkaline phosphatase, we studied the stability, kinetic properties, and commutability of separate preparations of the purified enzyme from human liver, intestine, bone, and placenta. The Michaelis constants (Km) for the preparations from liver, bone, and intestine agreed well with the Km values we obtained for five human serum specimens, whereas that for the placental isoenzyme differed significantly. The first three isoenzymes exhibited nearly identical response-surface patterns, which closely paralleled those observed for 12 human serum specimens (commutability), but not that of the placental isoenzyme. Thus, we believe that a reference material could equally well consist of either the bone, intestinal, or liver isoenzyme. All four isoenzymes were satisfactorily stable in temperature-accelerated degradation studies. We chose the liver isoenzyme as an appropriate reference material because liver tissue is easier to obtain than bone or intestine and the isoenzyme is abundant in liver, is easy to extract, and is the one most commonly increased in human serum. This material is stable at -20 degrees C, is free of interfering and degradative enzymes and, being of human origin, is commutable with the enzyme in human serum.

Some pitfalls in the quality control of alkaline phosphatase activity.

A study of the pH optimum of alkaline phosphatase activity in calibration and control serum preparations reveals a wide variation in pH optima. It is shown that as small an error of +/- 0.05 pH units in preparation of the buffered substrate results in an error in the order of +/- 5% in the response between the calibrating serum and patient specimens. It is recommended that a calibration serum should closely parallel the pH optimum of alkaline phosphatase in human sera and that a quality control serum containing alkaline phosphatase of alternative source to that present in the calibration serum be included with patient samples as a sensitive means of monitoring pH changes in the buffered substrate.

An assessment of the performance of the SMA 12/micro autoanalyser in clinical use.

The performance of the SMA 12/micro autoanalyser in clinical use in a children's hospital is described. Details of the precision and accuracy over a period of 134 operating days are given, and some of the factors which influence the results are discussed. The slow operating speed and analytical difficulties on the sodium, potassium, and cholesterol channels made the analyser unsuitable for routine use in this laboratory.

Standardization of the alkaline phosphatase determination with human placental phosphatase.

The influence of changes in temperature and salt concentrations on the enzymic activity of 3 different alkaline phosphatase preparations has been examined. Beef liver phosphatase is labile; the activity is already lost by lyophilization. Hog intestinal and especially human placental phosphatase are more stable. In contrast to reconstituted sera no preincubation is necessary to restore the enzymic activity. A lyophilized human placental phosphatase, kept at room temperature, showed no change in activity in a period of about one year. A mean value of enzymic activity of 133 U/l was observed with a coefficient of variation of 2.4% for 12 determinations. It is suggested to assign a value to such a preparation and to use it as a primary standard in the determination of the alkaline phosphatase.