Patrick F. Fottrell

Osteocalcin: Diagnostic Methods and Clinical Applications

Osteocalcin is a small (Mr 5800), very interesting bone specific protein, synthesized by osteoblasts and measured in plasma as a biochemical indicator of bone formation. Many immunoassays for osteocalcin have been developed, including radio- and enzymoimmunoassays, with the use of monoclonal and polyclonal antibodies. These are used in many different clinical settings, including bone, kidney, and liver diseases. However, there is a wide range of published values for plasma osteocalcin concentrations in control and patient samples and this has hindered a more widespread adoption of osteocalcin measurement by clinicians. This review discusses how various immunoassays for osteocalcin may contribute to the wide variation of published values and suggests approaches for the development of standardized assays. For example, epitope specificity and immunoreactivity with multiple forms of osteocalcin and osteocalcin peptides in plasma are discussed. It also includes a recent update on interesting clinical applications of osteocalcin.

Purification and characterization of aspartate aminotransferases from soybean root nodules and rhizobium japonicum

Abstract At least four electrophoretically distinct forms of aspartate aminotransferase were detected in root nodules of soybeans ( Glycine max L.). Two forms originated from the cytosol of the host plant, a third from the mitochondria of the host and a fourth from the bacterial component ( Rhizobium japonicum ). The properties of aspartate aminotransferase purified from nodule cytosol and from R. japonicum were compared. Both aminotransferases had many similar features including mol. wt., pH optimum and mode of action. However, the cytosol enzyme was much more resistant to a wide variety of inhibitors. Possible roles of the transaminases in ammonia assimilation in nodules is discussed.

Subcellular localization of enzymes involved in the assimilation of Ammonia by soybean root nodules

Abstract Ammonia, the primary product of nitrogen fixation is rapidly incorporated into a number of amino acids such as glutamate and aspartate. A novel enzyme system glutamine: 2-oxoglutarate aminotransferase oxidoreductase, which probably has an important role in ammonia assimilation has been detected, in the present studies, in the rhizobial fraction of soybean root nodules and in Rhizobium japonicum grown in culture. The role of this latter enzyme and other enzymes such as glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in ammonia assimilation by soybean nodules is discussed.

Interactions between an unusual aspartate aminotransferase from Rhizobium japonicum and pyridoxal-5′-phosphate studied by affinity chromatography

Aspartate aminotransferase (AAT)(L-aspartate; 2-oxoglutarate aminotransferase, EC 2.6.1 .l) from plant and mammalian sources are often resolved i.e. separated from their cofactor, pyridoxal-5’-phosphate (PLP) with some difficulty [ 1,2] . In contrast, AAT from some Rhizobium bacteria is very easily resolved [3] and, as shown in the present paper, is therefore amenable to purification by affinity chromatography using PLP as ligand. This report describes interactions between PLP and AAT from Rhizobium japonicum, studied by an affinity chromatography system which is easier to prepare than the one recently described by Collier and Kohlhaw [4]. The results demonstrate the effectiveness of affinity chromatography both in the purification of apo-transaminases and in the study of coenzyme-apoenzyme interactions.

A study of some enzymes and isoenzymes of carbohydrate metabolism in human endometrium during the menstrual cycle

Abstract The levels of seven enzymes were measured in endometrium from 208 women all of whom had regular cycles with normal menstrual losses. The activity of all the enzymes increased during the secretory phase and the increases were reflected in some instances by the appearance of additional isoenzymes on starch gel electrophoresis patterns.

Purification and properties of pyruvate kinase from human lung

Abstract Pyruvate kinase was purified to homogeneity from human lung and characterised kinetically. The enzyme has a molecular weight of 250 000 and a subunit molecular weight of 62 000. An amino acid analysis of the pyruvate kinase followed by peptide mapping of a tryptic digest confirmed that the enzyme contained 4 identical subunits.

The Clinical Usefulness of Salivary Progesterone Measurement for the Evaluation of the Corpus luteum Function

The present study was designed to construct reliable daily salivary progesterone profiles throughout the luteal phase to accurately evaluate the corpus luteum function. Furthermore, we investigated the clinical relevance of a simple midluteal salivary progesterone estimation for the diagnosis of luteal phase insufficiency by determining the diagnostic efficiency and cutoff values. A total of 121 women were divided into 3 groups; normal luteal function, luteal phase insufficiency and unclassified group, based on basal body temperature recordings and serum progesterone levels at 3 sampling points during the midluteal phase. Salivary progesterone values across the luteal phase of the normal luteal function group were significantly increased from day 1 to day 4, remained constant from day 5 to day 9 (mean ± SD, 318 ± 170 pmol/l on day 5, 287 ± 169 pmol/l on day 9; urinary LH surge = day 0) and decreased thereafter. Salivary progesterone concentrations in the luteal phase insufficiency group showed significantly lower values compared with those in the normal group between days 3 and 10. The cutoff values of 189 pmol/l in the midluteal phase yielded a sensitivity of 78.0% and a specificity of 76.5%. Our results suggest that daily salivary progesterone profiles during the luteal phase and a simple estimation of midluteal salivary progesterone appeared to be useful for the diagnosis of luteal phase defects.

Low-dose progesterone therapy in oestrogenised postmenopausal women: effects on plasma lipids, lipoproteins and liver function parameters

BACKGROUND Cardiovascular disease among older women is a major health problem and is the leading cause of death in this group in developed countries. The risk is reduced in oestrogen users secondary to favourable lipid changes, but the beneficial effect of oestrogen may be counteracted when concomitant progestogens are administered. OBJECTIVE To study the effects of a novel hormone replacement therapy regimen on liver enzymes, lipids and lipoproteins in postmenopausal women. DESIGN Prospective open, non-comparative trial for 12 months. METHODS 40 healthy postmenopausal women, (mean age +/- S.D.), 53.5 +/- 3 years received 0.625 mg of conjugated equine oestrogen daily and 100 mg of micronised oral progesterone (P) for the first 23 days every calendar month for 12 months without interruption. MAIN OUTCOME MEASURE Gonadotrophins, liver function parameters and lipoproteins were measured before treatment and at the 6th, 9th and 12th months of treatment. RESULTS Compliance with treatment was confirmed by a 33% decrease in mean serum level of follicle stimulating hormone at the end of 1 year of treatment. In the same period, the mean serum cholesterol, LDL and LDL/HDL ratio decreased by 6%, 16% and 23% of the base line levels, respectively. The percentage changes in triglycerides and HDL from the basal levels were +32% (P < 0.001) and +15% (P < 0.05), respectively. CONCLUSION These results indicate that near continuous administration of fixed low-dose of P has no adverse effects on the lipid milieu of postmenopausal women when combined with long-term continuous oestrogen replacement therapy provided women with borderline triglyceridaemia are excluded.

Similarities between pyruvate kinase from human placenta and tumours.

Pyruvate kinase (PyK) (EC 2.7.1.40) which catalyses one of the three physiologically irreversible steps in glyolysis has been studied in animals such as rat where it occurs in three different isoenzymic forms [1 ]. Rat liver has two PyK isoenzymes one form termed type L is under hormonal and dietary regulation whereas the other form type M 2 is not and more closely resembles the third PyK isoenzyme, type M 1 which is present in muscle and brain. The three types of PyK have distinct kinetic properties and react differently with several metabolites such as ATP, fructose 1, 6-diphosphate and amino acids. For instance, L type PyK is activated by fructose 1,6-diphosphate and inhibited by ATP whereas type M 1 is not. In this paper we describe the purification from human placenta of a distinct PyK isoenzyme with properties different from PyK in human liver and muscle. The placental isoenzyme of PyK has a number of features in common with PyK from some human tumours.

Purification and characterization of an aminoacyl proline hydrolase from guinea-pig intestinal mucosa.

The purification of an aminoacylproline hydrolase from guinea-pig intestinal mucosa is described. The enzyme, which is an aminopeptidase has a molecular weight of 112 000 and is activated by manganese and inhibited by zinc. Unlike other aminoacylproline hydrolases this enzyme displayed a broad substrate specificity. However, it was preferentially active against dipeptides containing proline in the C-terminal position.