The stability in vitro of bioactive and immunoreactive LH in human blood and plasma

1988 ◽  
Vol 117 (1) ◽  
pp. 139-145 ◽  
Author(s):  
A. Tsatsoulis ◽  
K. Mavroudis ◽  
J. Frost ◽  
A. Lambert ◽  
S. M. Shalet ◽  
...  
Keyword(s):  
Human Blood ◽  
Prolonged Storage ◽  
Human Blood Plasma ◽  
Immunological Activity ◽  
Human Pituitary ◽  
Reference Preparation ◽  
Degree Of Stability ◽  
The Stability ◽  
Mouse Leydig Cell

ABSTRACT The degree of stability in vitro of bioactive and immunoreactive LH in human blood, plasma and serum was examined. Bioactivity and immunoreactivity of LH were assayed by the dispersed mouse Leydig cell assay and by standard radioimmunoassay respectively, using the same reference preparation (first international reference preparation for human pituitary LH 68/40 for immunoassay). Bioactive and immunoreactive LH were stable in blood and plasma at 22 °C for up to 4 and 24 h respectively, and in blood at 4 °C for up to 24 h. There was no loss of biological or immunological LH activity in plasma which had been either snap-frozen and stored at −70 °C, allowed to freeze at −20 °C and stored at that temperature or kept at 4 °C for 24 h and then stored at − 70 °C. Likewise, the levels of LH in plasma and serum which had been stored at either − 20 or − 70 °C and then thawed and refrozen up to four times remained unchanged. In addition, the biological and immunological activity of LH was not affected after vortexing samples of plasma or serum for up to 60 s. Bioactive LH was also stable in plasma samples after prolonged storage (up to 9 months) at either −70 or −20 °C. We conclude that LH bioactivity and immunoreactivity are stable in blood and plasma following a variety of treatments commonly experienced during normal handling of a blood sample after venepuncture. J. Endocr. (1988) 117, 139–145

scholarly journals An In Vitro Analysis of the Effects of Intravenous Lipid Emulsion on Free and Total Local Anaesthetic Concentrations in Human Blood and Plasma

2014 ◽  
Vol 2014 ◽  
pp. 1-7
Author(s):  
Louise Ann Clark ◽  
Jochen Beyer ◽  
Andis Graudins
Keyword(s):  
Blood Plasma ◽  
Human Blood ◽  
Local Anaesthetic ◽  
Whole Blood ◽  
Lipid Emulsion ◽  
Equilibrium Dialysis ◽  
Human Blood Plasma ◽  
Intravenous Lipid Emulsion ◽  
Vitro Effect

Background. Intravenous lipid emulsion (ILE) is recommended as a “rescue” treatment for local anaesthetic (LA) toxicity. A purported mechanism of action suggests that lipophilic LAs are sequestered into an intravascular “lipid-sink,” thus reducing free drug concentration. There is limited data available correlating the effects of ILE on LAs.Aims. To compare the in vitro effect of ILE on LA concentrations in human blood/plasma and to correlate this reduction to LA lipophilicity.Method. One of four LAs (bupivacaine-most lipophilic-4 mg/L, ropivacaine-6 mg/L, lignocaine-14 mg/L, and prilocaine-least lipophilic-7 mg/L) was spiked into plasma or whole blood. ILE or control-buffer was added. Plasma was centrifuged to separate ILE and total-LA concentration assayed from the lipid-free fraction. Whole blood underwent equilibrium dialysis and free-LA concentration was measured. Percent reduction in LA concentration from control was compared between the LAs and correlated with lipophilicity.Results. ILE caused a significant reduction in total and free bupivacaine concentration compared with the other LAs. Ropivacaine had the least reduction in concentration, despite a lipophilicity similar to bupivacaine. The reduction in LA concentration correlated to increasing lipophilicity when ropivacaine was excluded from analysis.Conclusion. In this first in vitro model assessing both free- and total-LA concentrations exposed to ILE in human blood/plasma, ILE effect was linearly correlated with increasing lipophilicity for all but ropivacaine.

A COMPARISON OF PREPARATIONS OF HIGHLY PURIFIED HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE: DIFFERENCES IN THE FOLLICLE-STIMULATING HORMONE POTENCIES AS DETERMINED BY IN-VIVO BIOASSAY, IN-VITRO BIOASSAY AND IMMUNOASSAY

1981 ◽  
Vol 91 (2) ◽  
pp. 353-362 ◽  
Author(s):  
P. L. STORRING ◽  
A. A. ZAIDI ◽  
Y. G. MISTRY ◽  
BERIT FRÖYSA ◽  
BRIDGET E. STENNING ◽  
...  
Keyword(s):  
Biological Activity ◽  
Follicle Stimulating Hormone ◽  
In Vitro Bioassay ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Potency ◽  
Reference Preparation ◽  
In Vivo Bioassay

The FSH potencies of 12 preparations of highly purified human pituitary FSH, originating from six different laboratories, were determined by in-vivo and in-vitro bioassays and by immunoassay in terms of the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (IRP; coded 69/104). The contamination of these FSH preparations with LH was also determined. Estimates of protein content were based on the absorbance at 280 nm of solutions of the preparations, assuming that A1%1 cm 280 = 10. The FSH potencies varied between different preparations from 827 i.u./mg to 13 100 i.u./mg by in-vivo bioassay; from 2930 to 14 600 i.u./mg by in-vitro bioassay and from 1680 to 5690 i.u./mg by immunoassay. The ratios of in-vivo biological activity relative to in-vitro biological activity and to immunoreactivity respectively varied between preparations from 0·06 to 2·3 and from 0·15 to 4·1, and there was a significant positive correlation between each of these ratios and the in-vivo biological potency of the preparations; such differences could be due to varying degrees of sialylation between preparations. On the other hand the ratios of in-vitro biological activity to immunoreactivity between preparations were fairly constant (approx. 2). The excess biological activity relative to immunoreactivity observed, in terms of the IRP, in all these materials is consistent with recent findings of some immunoreactive FSH in the IRP unassociated with biological activity. These data did not demonstrate any significant advantage, in terms of FSH in-vivo biological potency, from the use of fresh-frozen rather than acetone-dried pituitary glands for the isolation of FSH. Contamination of all these preparations with LH appeared to be less than 3% (w/w), as determined by in-vitro bioassay and by immunoassay. The results of this study are discussed in relation to the selection of material for an international reference preparation for immunoassay and attention is drawn to the value of high in-vivo biological FSH potency as a criterion of the identity of a preparation as well as of its freedom from contaminants without FSH biological activity.

Action of heparin and heparin-precipitated fraction of human blood plasma on antibody-producing cells in vitro

1976 ◽  
Vol 81 (1) ◽  
pp. 70-72
Author(s):  
S. V. Kaznacheev ◽  
V. A. Kozlov ◽  
E. M. Petrova ◽  
V. P. Lozovoi
Keyword(s):  
Blood Plasma ◽  
Human Blood ◽  
Human Blood Plasma

THE INTERNATIONAL REFERENCE PREPARATION OF HUMAN PITUITARY LUTEINIZING HORMONE, FOR IMMUNOASSAY

1978 ◽  
Vol 88 (2) ◽  
pp. 250-259 ◽  
Author(s):  
P. L. Storring ◽  
D. R. Bangham ◽  
P. Mary Cotes ◽  
Rose E. Gaines ◽  
S. L. Jeffcoate
Keyword(s):  
Luteinizing Hormone ◽  
Reproductive Organ ◽  
Expert Committee ◽  
Confidence Limits ◽  
Binding Assays ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Standardization ◽  
Reference Preparation

ABSTRACT The preparation and nature of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay are described. A collaborative assay of this material (coded 68/40) in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH (ICSH)), for Bioassay (coded 69/104) was carried out by twelve laboratories in seven different countries, using different bioassay and immunoassay methods. The weighted combined potency estimate (with 95 % confidence limits) was 52.1 (46.3–58.7) IU/ampoule with male accessory reproductive organ weight gain assays; 80.2 (73.2–87.9) IU/ampoule with ovarian ascorbate depletion assays; 127 (124–129) IU/ampoule with in vitro Leydig cell testosterone production assays; and 124 (121–126) IU/ampoule with testis receptor binding assays. Immunoassay estimates in terms of the same standard were heterogeneous and gave an unweighted mean potency estimate of 33.2 IU with 95% confidence limits of 14.8– 74.4 IU/ampoule. Estimates from different methods gave significantly different results, and the reasons for this are discussed in terms of the differences between the materials being compared and the methods used in the comparison. These data illustrate the conceptual difficulties involved in comparing hetero geneous reference preparations, especially by both bioassay and immunoassay, and some of the causes of inevitable discontinuity of assay results, as described in the 26th Report of the WHO Expert Committee on Biological Standardization. On the basis of these results, and in the interest of maintaining continuity of its unitage, the International Reference Preparation has been allocated a potency of 77 IU/ampoule.

BIOLOGICAL AND IMMUNOLOGICAL PROPERTIES OF DIFFERENT MOLECULAR SPECIES OF HUMAN FOLLICLE-STIMULATING HORMONE: ELECTROFOCUSING PROFILES OF EIGHT HIGHLY PURIFIED PREPARATIONS

1982 ◽  
Vol 92 (2) ◽  
pp. 195-204 ◽  
Author(s):  
A. A. ZAIDI ◽  
B. FRÖYSA ◽  
E. DICZFALUSY
Keyword(s):  
Biological Activity ◽  
Sucrose Density Gradient ◽  
Relative Distribution ◽  
Human Pituitary ◽  
Sialic Acid Content ◽  
Reference Preparation ◽  
Proportional Increase ◽  
Α And Β Subunits ◽  
Ph Range

Eight highly purified human pituitary FSH preparations and purified preparations of the α-and β-subunits of FSH were fractionated by an electrofocusing technique in the pH range of 2·5–10·0 on a sucrose density gradient. The human (h) FSH activity in each of the eluted fractions was monitored by an in-vitro bioassay and a radioimmunoassay procedure. After electrofocusing, the overall recovery of the biological activity of the eight preparations was between 80 and 94% (mean 88%). On the other hand, the recovery of immunoreactivity ranged between 30 and 84% (mean 71%). A loss of over 85% of hFSH immunoreactivity was observed when the α- and β-subunits of hFSH were fractionated by the same procedure. The specific loss of varying amounts of immunoreactivity in all preparations during electrofocusing was also reflected by a proportional increase in the ratios of biological activity (B) to immunoreactivity (I); preparation A, which exhibited a loss of 70% of the immunoreactivity, had a threefold increase in its B/I ratio after electrofocusing. Significant differences were observed in the electrofocusing profiles of the eight preparations both in terms of their pI values and of their spread. The disparity in the relative distribution of hFSH activities in different pH regions suggested major differences in the carbohydrate moieties (sialic acid content) of the preparations studied, probably as a result of the chemical manipulations involved in the purification of the hormone. It is suggested that a combination of several (but certainly not all) of the preparations might serve as a provisional International Reference Preparation for hFSH radioimmunoassays.

A Novel Validation of Analysis Method for Determining Nicotine Levels in Human Blood Plasma (In vitro) High-Pressure Liquid Chromatography Ultraviolet Detector

2020 ◽  
Vol 10 (02) ◽  
pp. 238-243
Author(s):  
Ghassaq T. Al-Ubaidi ◽  
Ahmed A. Abbas ◽  
Ali A. Taha ◽  
Qasim S. Sharhan
Keyword(s):  
Liquid Chromatography ◽  
Blood Plasma ◽  
Human Blood ◽  
Analytical Methods ◽  
Limit Of Detection ◽  
Uv Detection ◽  
Human Blood Plasma ◽  
Analysis Method ◽  
System Suitability

The necessity of nicotine analysis in blood plasma is increasing along with the increased number of smokers and nicotine poisoning cases. One of the analytical methods for nicotine is using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detector because it has been commonly owned by instance in Indonesia. To guarantee accuracy, an analytical method can be used, and it must be validated. This research was the purpose of finding out the validity of the nicotine analysis method in human blood plasma (in vitro) using HPLC with UV detection. Blood plasma samples remained treated with centrifugation procedure by protein denaturation method using acetonitrile. The compounds were analyzed using methanol and buffer acetate 0.01 M (pH 5) 85:15 v/v as a mobile phase on an octadecylsilane column 250 mm, with UV detection at 254 and 260 nm, and flow rate 0.6 mL/minute. Parameter of analytical methods that were validated includes selectivity, accuracy, precision, repeatability, linearity, limit of detection (LoD), limit of quantification (LoQ), and system suitability. According to the result, the selectivity was 2.479, repeatability expressed by its variation coefficient = 0.701%, linearity at range 5–22 μg/mL expressed by coefficient correlation (r) = 0.996. Based on the chromatogram’s area under a curve, the LoD value was found 2.021 μg/mL, LoQ value was 6.737 μg/mL, the accurate percentage was 112.49 to 114.12%, and precision (% CV) was 2.15 to 3.95%. The system suitability from retention time and chromatogram’s area under curve showed % CV 0.70 and 1.64%. According to the experiment result, all parameters meet the requirements of validation criteria.

Effect of x-ray contrast agents on human blood plasma interleukin-2 level in vitro

1992 ◽  
Vol 114 (3) ◽  
pp. 1315-1317
Author(s):  
Yu. K. Napolov ◽  
N. U. Borsukova ◽  
N. L. Shimanovskii
Keyword(s):  
Blood Plasma ◽  
Contrast Agents ◽  
Human Blood ◽  
Interleukin 2 ◽  
Human Blood Plasma ◽  
X Ray

A SENSITIVE AND SPECIFIC IN VITRO BIOASSAY METHOD FOR THE MEASUREMENT OF FOLLICLE-STIMULATING HORMONE ACTIVITY

1979 ◽  
Vol 91 (2) ◽  
pp. 224-237 ◽  
Author(s):  
M.-P. Van Damme ◽  
D. M. Robertson ◽  
R. Marana ◽  
E. M. Ritzén ◽  
E. Diczfalusy
Keyword(s):  
Human Growth ◽  
Assay Method ◽  
Human Pituitary ◽  
Bioassay Method ◽  
Specific Radioimmunoassay ◽  
Reference Preparation ◽  
Low Levels ◽  
Lh Rh ◽  
Dose Levels

ABSTRACT An in vitro bioassay method for hFSH is presented. The method is based on the principles previously described by Dorrington et al. (1976b) and involves the assay of oestradiol produced from 19-hydroxyandrostenedione by dispersed Sertoli cells of 10-day old rats when cultured in the presence of graded doses of FSH. Using the 1st International Reference Preparation for human pituitary gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104) as standard, the useful range of the method is from 0.5 to 32 mIU/chamber (2 to 128 mIU/ml). The sensitivity of the method is 0.5 mIU/chamber. The mean index of precision <UNK> obtained from 16 multiple assays over 2 or 3 dose levels was 0.084. Parallelism was obtained between the 69/104 preparation and all preparations under study. The practicability of the proposed assay method is such that 15 preparations at 3 dose levels can be assayed by one person in 3 days. The specificity of the assay was investigated by determining the FSH activity in the following preparations: hFSHa-α and β-subunits, hLH, hCG, hTSH, ACTH, human growth hormone (hGH) human prolactin (hPRL) and luteinizing hormone-releasing hormone (LH-RH). The ACTH, hGH, hPRL and LH-RH preparations studied showed no detectable FSH activity in the assay. In the remaining preparations very low levels of FSH activity were found, corresponding to 0.004 to 0.6 % of the weight of these preparations when compared with a highly purified hFSH preparation, suggesting that the method is specific for FSH. The possible synergistic or antagonistic influence of the above preparations when assayed in the presence of the 69/104 preparation was also assessed. No evidence of a synergistic or antogonistic effect was found. The assay of the hFSH potencies of a limited number of hFSH preparations of varying purity by the proposed in vitro bioassay, an hFSH radioreceptor method and an hFSH specific radioimmunoassay technique revealed that – although the relationship of the various potencies obtained with each method showed a close agreement – the bioassays yielded the highest potency estimates, and the radioimmunoassays the lowest ones. Since the proposed bioassay method is sensitive and considered to be specific for hFSH activity, it provides a suitable basis for the assessment of the specificity of other in vitro methods (radioreceptor and radioimmunoassay) currently used for detecting low levels of FSH activity.

EFFECT OF STORAGE ON THE BIOACTIVITY OF TWO INTERNATIONAL REFERENCE PREPARATIONS OF HUMAN PITUITARY LUTEINIZING HORMONE IN VITRO

1979 ◽  
Vol 81 (1) ◽  
pp. 153-155 ◽  
Author(s):  
D. M. ROBERTSON ◽  
BERIT FRÖYSA
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Interstitial Cell ◽  
Saline Solution ◽  
Plasma Albumin ◽  
Human Pituitary ◽  
International Reference ◽  
Bovine Plasma ◽  
Reference Preparation

There was no loss of biological activity in vitro when the 1st International Reference Preparation (IRP) for human pituitary gonadotrophins [FSH and LH/interstitial cell stimulating hormone (ICSH) for bioassay] code no. 69/104 and the 1st IRP for human pituitary LH/ICSH [for immunoassay] code no. 68/40 were stored for 1 year at −70 °C in a buffered 0·8% saline solution containing 1% bovine plasma albumin (BPA). However, storage of the 69/104 preparation at −20 °C in either 0·1 or 1% BPA, or at −70 °C in the presence of 0·1% BPA showed a small but significant decrease (∼ 10%) in activity over the same period. It is, therefore, advantageous to store these reference preparations at −70 °C in the presence of 1% BPA.

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