M. Worsfold

Enzyme detection using phenazine methosulphate and tetrazolium salts: Interference by oxygen

Abstract Phenazine methosulphate (PMS) is used widely as an intermediate electron carrier for coupling the production of NADH or NADPH to the reduction of tetrazolium salts to coloured formazans. Nitro-blue tetrazolium (NBT) is the most popular tetrazolium salt for this purpose, for both spectrophotometric assay of dehydrogenases and the visualisation of dehydrogenases after electrophoresis or in tissue sections. It has been widely assumed (1) that oxygen does not interfere with this reaction, even though it is known that reduced PMS reacts very readily with oxygen (2–4). This assumption has been supported in a study by Altman (5). Sowerby and Ottaway (6) observed that oxygen reduced the yield of formazan in their assay for glutamine, but they failed to detect its consumption during the reaction and postulated other reasons for their nonquantitative results. Oxygen always appears to be ignored when this reaction is used to stain enzyme zones in histochemistry or after electrophoresis. We report here that atmospheric oxygen interferes seriously with this reaction under a wide range of conditions, especially at low rates of reduced pyridine nucleotide production.

Superoxide dismutase: A direct, continuous linear assay using the oxygen electrode

Abstract A continuous method for the assay of superoxide dismutase activity which gives a linear recording of the reaction whose slope is a linear function of the amount of enzyme added is described. An oxygen electrode is used to measure the consumption of oxygen which results from the photochemical generation of superoxide from dissolved oxygen followed by its dismutation to oxygen and hydrogen peroxide. Excess superoxide is scavenged by a tetrazolium salt which oxidizes it back to oxygen.

Serum osteocalcin and other indices of bone formation: An 8-decade population study in healthy men and women

In a study of 435 healthy men and women aged 17-97 yr, serum osteocalcin and alkaline phosphatase were measured together with 99TcMDP retention in women. In women, serum osteocalcin falls to a nadir at 35-39 yr, and the mean then rapidly rises 2-fold to a plateau from 50-75 yr. 99TcMDP retention falls to a minimum at 40-45 yr and then rises steadily with increasing age. Serum alkaline phosphatase rises in an indeterminate fashion from 20-25 yr onwards. Osteocalcin in men fell until age 60-70 yr and hardly changed thereafter, whereas serum alkaline phosphatase reached a minimum at age 30-40 yr and thereafter rose with age, as in women.

Knee angles and rickets in Nigerian children

Knee angles of 2,036 normal Nigerian children up to 12 years old were measured directly or from photographs. The knees were bowed (varus) in the first 6 months. At 21 to 23 months, the distribution of angles became strongly bimodal: about half were varus and half were valgus (knock-kneed), with few in between. After this they were all valgus, with few exceptions. Hence, the change from varus to valgus in individual infants must be sudden (a few weeks), although the changeover of the whole population appears smooth and gradual. They became maximally and uniformly knock-kneed (−7.1° ± 1.4°) between 3 and 3.5 years, with little change thereafter. On the other hand, 120 patients with rickets discovered during screening had large knee angles, in either sense, with a bimodal distribution and frequency maxima at +10° (varus) and −12° (valgus). Varus knee is uncommon after 2 years. Large knee angles between 2 and 5 years suggest rickets.

Plasma aluminium in a reference population: the effects of antacid consumption and its influence on biochemical indices of bone formation

Abstract. Aluminium is involved in the etiology of several complications of chronic renal failure and has been firmly established as having toxic effects on bone tissue. We have measured plasma aluminium together with serum osteocalcin, procollagen I C‐terminal peptide and total alkaline phosphatase activity in healthy subjects and in a group of subjects who consumed aluminium‐containing and non‐aluminium containing antacid preparations, with normal renal function. Age‐related healthy reference ranges for plasma aluminium are presented and the effects of chronic antacid consumption on plasma aluminium and biochemical markers of bone formation investi gated.

Analytical micro-preparative electrophoresis: Quantitation of phosphoglucose isomerase isoenzymes

A simple micro-preparative electrophoresis apparatus is described using a thin vertical slab gel and continuous elution through a narrow horizontal slot. Phosphoglucose isomerase (PGI) isoenzymes in lysates of 0.2 to 100 microliters of erythrocytes were separated using this system and their activities determined using an on-line, air-segmented, continuous flow assay. Ninety-five percent recovery of the separated isoenzymes after electrophoresis was achieved and the factors influencing recovery of PGI and hemoglobin were investigated. Thus, quantitative recovery has been achieved without any loss of resolution. Up to 29 successive separations of PGI isoenzymes have been carried out on a single gel.