Seasonal variation of vitamin D3 and 25-hydroxy vitamin D3 in human serum

High levels of triiodothyronine have been found in a patient with Graves' disease caused by circulating antibodies able to bind specifically with serum triiodothyronine. High values found were due to the interference of the endogenous antibody with the conventional techniques of radioimmunoassay used for the evaluation of the hormone. An analytical pattern to identify these antitriiodothyronine antibodies is described.     

A specific assay of vitamin D-3 in human serum.

A specific mass fragmentographic method for determination of free vitamin D-3 in biological fluids has been developed. The method involves addition of vitamin D-2 to the sample, followed by freeze-drying and extraction with Chloroform/methanol. The amount of vitamin D-2 added should be more than a hundred-fold the amount of vitamin D-3 expected in the sample. The vitamin D and previtamin D present in the extract are heat-equilibrated with each other and then subjected to thin-layer chromatography. Under the Chromatographie conditions employed, there is an only partial separation between vitamin D-2 and vitamin D-3. The material in the appropriate Chromatographie zone is converted into trimethylsilyl ether and analyzed by gas chromatography-mass spectrometry with use of a multiple ion detector. The amount of vitamin D-3 is determined from the ratio between the recordings at m/e 325 and m/e 337. The two ions used correspond to the M — 131 peak in the mass spectrum of trimethylsilyl ether of vitamin D-3 and vitamin D-2, respectively. The method was applied to the determination of vitamin D-3 in human serum and the accuracy of this application was ascertained by different recovery experiments. Considerably lower concentration of vitamin D-3 in serum was found with the present technique than with previous techniques. Thus analysis of serum from 19 healthy men and women gave values ranging from 0 to 6.3 ng/ml (mean about 2 ng/ml). There was little or no increase in concentration after shortterm irradiation of part of body area with UV light. Psoriatic patients subjected to treatment with UV light for 3–5 weeks, however, had considerably higher serum level of vitamin D-3 than normal. Administration of vitamin D-3 per os to one subject gave a transient increase in concentration of vitamin D-3 in serum with a peak value about 4 h after the administration.     

A quantitation of lactate dehydrogenase non-linearity.

It was the objective of this study to quantitative the non-linearity seen in the lactate dehydrogenase reaction and relate these findings to the wide variation between reported values from various sources. Data are based upon two sample groupings: 1200 routine samples and 200 selected elevated specimens rangingg from 200 to 2000 I.U. (LD-L). This sample population demonstrated a rate of activity decrease rangin from 2% to 13% per minute, dependent upon activity level.     

Serum 25-hydroxyvitamin D assay. Evaluation of chromatographic and non-chromatographic procedures

A competitive protein binding assay for serum 25-hydroxyvitamin D is described in which normal human serum is used as the source of binding protein. A serum sample is extracted with diethyl ether/methanol and then chromatographed using silica gel. Validation of the method is reported. Silica gel chromatography is compared with LH20 chromatography. The method is also compared with extraction techniques using diethyl ether and ethanol without subsequent chromatography. It is concluded that chromatography with either silica gel or LH20 is essential. The non-chromatographic methods investigated, in addition to giving much higher values than chromatographic methods, did not meet validation requirements with respect to accuracy and behaviour of samples on dilution.     

A simple method for the determination of free cortisol in plasma, with normal levels in men and women, including women taking low oestrogen contraceptives.

A small scale equilibrium dialysis method for the measurement of the non-protein bound cortisol in plasma is described. Dialysis is carried out in PTFE cells against an isosmolar Hepes buffer, equilibration occurring within 18 hours. Inter-assay variation was found to be approximately 6%. Samples taken from normal males and females and from oestrogen-treated females were analysed and the results compared with published levels. A significant difference in both the total and free cortisol levels was found between the normals and oestrogen-treated females.     

Binding of 3,5-diiodotyrosine to serum proteins.

The reversible binding of 3,5-diiodotyrosine (DIT) to human and bovine serum protein and to purified human serum prealbumin and human and bovine albumin has been studied by equilibrium dialysis. Maximum binding occurred at pH 8.6-9.0. Human serum bound DIT less than did bovine serum. Adult ox and fetal calf sera showed similar binding. The main DIT-binding protein of human serum was prealbumin. It showed a single affinity site with a Ka of 0.85 X 10(6) M-1 at pH 8.6 and 0.40 X 10(6) M-1 at pH 7.4. The affinity constant of serum albumin for DIT was 2.8 X 10(3) M-1 at pH 8.6. The elevated binding of DIT to bovine serum is essentially due to albumin whose affinity constant for DIT is 16-times higher than that of human serum albumin. Fetuin was not responsible for any noticeable DIT binding in fetal calf serum.     

Contributions of other sterols to the estimation of cholesterol.

The responses of 5alpha-cholestan-3beta-ol, 5alpha-cholest-7-ene-3beta-ol and cholesta-5,7-dien-3beta-ol, normally found in human serum, were examined by: (1) the Liebermann-Burchard reaction, (2) the Zak (ferric chloride) reaction, (3) an enzymatic cholesterol method monitored by estimating the amount of hydrogen peroxide produced, (4) an enzymatic cholesterol method monitored by observing the change in absorbance at 240 nm, and (5) gas chromatography. The results show that none of these methods is specific for cholesterol; contributions from the sterols examined range from zero to more than 150% relative to cholesterol. For the first four methods contributions depend on the conditions under which each test is performed.     

Arylamidases in normal and diseased human muscle.

Human skeletal muscle homogenates were found to contain enzymes that catalyze the hydrolysis of beta-naphthylamides of leucine, arginine and lysine, known substrates for neutral and basic arylamidases. They also contained a trace of activity towards alpha-aspartyl-beta-naphthylamide. The muscle arylamidases were found to be inhibited by p-chloromercuribenzoate, Hg2+ and puromycin. Leucyl, arginyl and lysysl arylamidases were slightly activated by cobalt ions. When compared to controls, no significant differences in muscle arylamidase activities were observed in patients with muscular dystrophies and certain denervating diseases.     

Application of nicotinamide-adenine dinucleotide analogs for clinical enzymology: a spectrophotometric method for clinical analysis of lactate dehydrogenase patterns with the use of a nicotinamide-adenine dinucleotide analog.

The activities of porcine lactate dehydrogenase (LHD) isoenzymes were analyzed using nicotinamide-adenine dinucleotide (NAD) or its analogs as a cofactor, with varying concentrations of L-lactate from 13 to 530 mM. The greatest differences between H4-type and M4-type isoenzymes in reaction rates were observed when their activities were compared in a reaction mixture containing 530 mM lactate and NAD, and also in a system of 13 mM lactate with thionicotinamide-hypoxanthine dinucleotide as a cofactor. The ratio of the LDH activity exerted in the former reaction mixture to that exerted in the latter was termed the N/T value. The N/T values of porcine H4 and M4 isoenzymes were 0.49 and 9.33, respectively. The N/T values of other three isoenzymes (H3M1, H2M2 and H1M3) were calculated by assuming that the single subunits H1 and M1 contribute one-fourth of the values of 0.49 and 9.33, respectively, and a given isoenzyme which is a combination of four subunits of H and M comprises the sum of their values. The calculated values agreed fairly well with the experimental results. The N/T value method was found to be applicable to human LDH isoenzymes, and sera from various patients were analyzed in comparison with hormonal sera. The method is particularly suitable for the numerical expression of LDH isoenzyme profiles.     

A simple and specific competitive protein binding assay for 24,25-dihydroxyvitamin D in human serum

A sensitive and specific competitive protein binding assay for 24,25-dihydroxyvitamin D was developed using rat kidney cytosol as the binding protein. It also included a chromatographic step on a Sephadex LH 20 column to separate the metabolite specifically. The average value for 24, 25-dihydroxyvitamin D in serum in 19 male and female healthy subjects aged from 20 to 40 was found to be 8.24 +/- 0.34 ng/ml (1.98 +/- 0.08 . 10(-8) mol/l) mean +/- S.D. In hepatic insufficiencies the levels are higher (14.15 +/- 3.59 ng/ml) than in normal subjects. A comparison between 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D levels was made.     

Isoelectrofocusing: a method of multiple applications for hemoglobin studies.

A microanalytical screening of hemoglobins by isoelectric focusing on acryl-amide gel columns is described. Addition of this technique to our regular analytical procedures revealed many mutants, praticularly some neutral ones which were not resolved by other conventional procedures. Extension of this technique to a quantitative scale permitted rapid isolation of some unstable mutants in an almost pure form ready for functional studies. The natural color of the separated components is often indicative of their nature and in cases like the sulfhemoglobin, Hbs M, they are of diagnostic value. The heme-losing, abnormal, unstable mutants, on the addition of external cyanhemin, take them up and change their isoelectric pH. This is an attractive visible procedure for their diagnosis. From all these results, it appears that isoelectric focusing as a single method brings more information than the other conventional procedures and hence can be considered as a semi-routine investigation for hemoglobin studies before deciding on a much longer procedure.     

Factors involved in the determination of triglycerides in serum: An international study

Ten laboratories analysed five different specimens in duplicate on ten separate occasions by one, or several, of five common triglyceride methods. Simple statistical data are presented and, as far as possible, these are interpreted in the light of the methods used and the results of chemical analyses of the materials. Great variability was found between the results of the participating laboratories. The major factor involved seems to be the material specific nature of the methods under study. At least one method in common use is contraindicated.     

Diagnosis of GM1 gangliosidosis based on detection of urinary oligosaccharides with high performance liquid chromatography

An improved, rapid, and sensitive method for the biochemical diagnosis of GM1 gangliosidosis based on the detection and quantification of urinary galactosyl-oligosaccharides with high performance liquid chromatography was developed. The oligosaccharides, in 50-100 microliters of urine, were converted to radioactively labeled oligosaccharide-alditols with NaB3H4 and fractionated on commercial silica-amine bonded, high performance liquid chromatography columns. Delineation between infantile, juvenile, and adult onset subtypes of GM1 gangliosidosis was possible by analysis of the levels of the excreted oligosaccharides and their characteristic elution profile. Infantile and juvenile patients contain identical numbers of oligosaccharide fractions (13 resolved components) but can be distinguished by 3-10-fold lower levels of oligosaccharides in juvenile patients and, in some cases by a disproportionately lower concentration of high molecular weight compounds. Adult onset patients were distinguished by substantially lower concentrations of urinary oligosaccharides, 130-180-fold below those in infantile patients, and the apparent absence of high molecular weight oligosaccharides.     

Incorporation of L-leucine and D-glucosamine into skin fibroblasts derived from cystic fibrosis and normal individuals

Cultured skin fibroblasts from patients with diagnosed cystic fibrosis were compared with those from normal individuals in respect to incorporation of l-leucine or d-glucosamine into the subcellular components, to uptake of α-isobutyric acid and calcium, and to (Na⁺-K⁺)ATPase activity, etc. No significant difference which could be attributed to the disease-specific basic metabolic abnormality was demonstrated. Cystic fibrosis skin fibroblasts may not express the primary genetic defect at the level of generalized transport mechanism and overall protein metabolism.     

A method for studying plasma transport of vitamin D applicable to hypervitaminosis D

In man, vitamin D is normally transported on a specific binding globulin (DBP) and on lipoproteins. In addition, binding to albumin occurs in the presence of vitamin D excess. Agarose gel electrophoresis was used to study the binding of radioactive vitamin D to plasma proteins in lipoprotein-free plasma (d > 1.21 g/ml). This method completely separates DBP from albumin and thus enables the quantification of vitamin D bound to these proteins in various clinical and experimental conditions. The same method can be used to study the transport of other vitamin D metabolites.     

A microfluorometric assay of leukocyte alpha-1,4-glucosidase.

We describe an improved method for detecting deficiency of the acid hydrolase, alpha-1,4-glucosidase in leukocytes, the enzyme defect in glycogen storage disease Type II (Pompe disease). The procedure requires smaller volumes of blood and less time than previous methods. The assay involves the separation of leukocytes by Peter's method for beta-glucosidase and a modification of Salafsky and Nadler's fluorometric method for alpha-glucosidase.     

Plasma somatomedin activity in vitamin A deficient children.

A role for vitamin A in the sulphation of mucopolysaccharides has been suggested. Somatomedin, a growth hormone dependent serum factor, has also been shown to stimulate the uptake of sulphate by cartilage. Therefore studies were undertaken on vitamin A deficient children to examine the possible interrelationship between vitamin A and somatomedin activity. Plasma somatomedin activity was markedly lowered in vitamin A deficient children and plasma HGH levels were in the normal range. The data suggest that vitamin A and somatomedin may be interrelated and also that plasma somatomedin activity may not always be determined by plasma growth hormone levels.     

A radioactive substrate and assay for alpha-L-iduronidase.

A novel radioactive substrate for alpha-L-iduronidase, anhydro[3H]mannitol-iduronide has been prepared. This compound is hydrolysed by alpha-L-iduronidase to yield anhydro[3H]mannitol, which can be separated from the substrate and counted. The assay, described for use with fibroblast homogenates, is satisfactory for the diagnosis of mucopolysaccharidosis I.     

Evaluation of a rapid, sensitive and specific assay for the determination of collagenolytic activity in biological samples.

Several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. A labelled collagen substrate was prepared using [3H]acetic anhydride. The specificity of the assay as well as conditions allowing an optimum detection limit were investigated. The influence of low temperatures, lyophilisation and salt concentration on Clostridium histolyticum collagenase have been investigated.     

Interaction of conjugated bile acids and detergents with a radiosorbent assay of vitamin B-12

The effect of conjugated bile acids and detergents on the radiosorbent technique for the determination of vitamin B-12 activity is reported. It is shown that whereas the non-ionic detergent Triton X-100 has no effect on the vitamin B-12-radiosorbent assay, the addition of ionic detergents (e.g. glycocholic acid, taurocholic acid or sodium lauryl sulfate) results in a falsely elevated vitamin B-12 activity presumably due to the disruption of the binding of vitamin B-12 to the intrinsic factor-Sephadex complex. This effect may be of importance not only to the radiosorbent assaying of vitamin B-12, but to the in vivo intestinal absorption of vitamin B-12 as well.