R. Heath

Plasma Amino Acids of the Mid-Trimester Human Fetus

The amino acid concentrations in umbilical cord plasma taken at fetoscopy were measured from 12 fetuses between 18 and 29 weeks gestation. Concurrent maternal plasma levels were measured. Fetal plasma showed consistently higher concentrations of plasma amino acids with the ratios varying between 1.1:1 and 3:1 and the difference always reached statistical significance. It is suggested that these results reflect the in vivo situation of the mid-trimester human fetus.

Hormonal Control of Carbonic Anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III (CA III) in the livers of adult male rats was found to be approximately 30 times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver CA III concentrations that could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomized female rats induced about a 5-fold increase in liver CA III concentration. Immunoprecipitational analysis of the products of liver mRNA translation in vitro with antiserum specific for CA III showed that hormonal control of the levels of CA III in rat liver is mediated by changes in the amount of translatable CA III mRNA. Marked changes in liver CA III concentrations were also observed in developing and aging male rats. Different control mechanisms appear to operate in mouse and man.

Radioimmunoassay of human muscle carbonic anhydrase III in dystrophic states.

A radioimmunoassay for the human isozyme carbonic anhydrase III (CAIII) has been developed. The assay can detect levels as low as 4 microgram/l of sample. Plasma CAIII levels in patients suffering from Duchenne muscular dystrophy were found to be up to 39 times greater than levels in a control group. Urine CAIII levels in patients suffering from Duchenne muscular dystrophy were not significantly different from the levels found in urine from normal adults. Measurement of plasma CAIII levels may be useful in prenatal diagnosis of Duchenne muscular dystrophy, and in investigation of adult skeletal muscle disease.

Carbonic anhydrase III in Duchenne muscular dystrophy

Plasma carbonic anhydrase III (CAIII) levels determined by radioimmunoassay have been compared, in detail, with creatine kinase (CK) as indices of Duchenne muscular dystrophy (DMD). CAIII levels were markedly elevated in all patients but variability of levels in a number of individual patients was higher than CK.

Carbonic anhydrase III in neuromuscular disorders

Plasma carbonic anhydrase III (CAIII) and creatine kinase (CK) were measured in 44 patients with a variety of neuromuscular disorders. Markedly elevated CK levels were associated with similarly increased levels of CAIII. In 9 patients, only the CAIII was elevated, but in 2 patients only the CK was raised. The determination of plasma CAIII is thus an important index of muscle disorder and is probably more sensitive than CK.

Red cells genetically deficient in carbonic anhydrase II have elevated levels of a carbonic anhydrase indistinguishable from muscle CA III.

Adult human red cells contain appreciable levels of two carbonic anhydrase (CA) isozymes, CA I ( I 1.6 ? 2.3 mg/g Hb) and CA I1 (1.8 ? 0.3 mg/g Hb), as determined by radioimmunoassay (RIA). Recently, biochemical and immunological studies have demonstrated the presence of an additional red cell carbonic anhydrase with properties indistinguishable from skeletal muscle CA III.2*3 It is present at levels of 147 ? 17 pg/g Hb assuming that the RIA, set up for the skeletal muscle CA 111,4 is in fact detecting a CA isozyme in red cells immunologically indistinguishable from muscle CA 111. Purification of this red cell “CA 111” by the methods used for isolating skeletal muscle CA I11 (affinity chromatography and gel f i l t ra t i~n)~ has, however, resulted in yields of less than 10% of that e ~ p e c t e d . ~ The purified red cell “CA 111” and skeletal muscle CA I11 have identical elution properties from reverse-phase pBondapak CIS high performance liquid chromatography (HPLC) column^^-^ and preliminary peptide-map data indicate that the allelic polymorphism found in skeletal muscle CA I11 (31 Ile -+ Val)5 is also present in the “CA 111” purified from outdated red cells pooled from several individuals. These data and the finding that the characteristic electrophoretic mobilities of skeletal muscle CA I11 from different mammals are mimicked by their red cell “CA 111” counterparts strongly support the view that we are examining products of the same gene. Future work at the gene level should confirm this. However, we must take into account that the phenomenon of gene conversion, whereby adjacent homologous genes can evolve “in concert” (e.g. , a globin and y globin gene^)^; this provides a basis for the possibility that there are two tissue-specific CA I11 genes encoding identical or almost identical proteins. The CA I and CA I1 genes are closely linked on chromosome 3 of the mouse.8 There is good evidence for such linkage in the Old World monkey, Macaca nerne~trina,~ and other mammals.1° It seems likely that CA I and CA I1 will also be linked in humans although only CA II has been mapped (to chromosome 8) so

Fetal Plasma Carbonic Anhydrase III and Creatine Kinase in Duchenne Dystrophy

Raised levels of creatine kinase (CK) and carbonic anhydrase 111 (CA 111) have been found in the plasma of patients with Duchenne muscular dystrophy (DMD) and there is good correlation between them. CA 111 is also virtually specific to skeletal muscle where it is induced quite early in gestation. These factors suggest the potential usefulness of measuring CA I11 and CK in fetal plasma for prenatal diagnosis of DMD. This was investigated as follows. About 80 individual fetal plasma samples were obtained by fetoscopy from pregnancies not at risk for muscle disorders. Sampling was performed at 18-21 weeks gestation. Particular care was taken in all sample collection and handling procedures to ensure pure fetal blood samples were obtained. Radioimmunoassay of CA I11 and spectrophotometric assay of CK enabled control distributions to be calculated. Upper normal limits were fixed at the 95th percentile for both CK and CA I11 (e.g., for CA 111 see FIG. 1). Eleven at risk fetuses were investigated for CA 111. These were divided into low and high risk groups. All three of the low risk fetuses gave CA 111 values below the 95th percentile and normal babies were born at term. All of the high risk fetuses were terminated. From the combined carrier risk of the mothers, three affected fetuses were predicted in this group. In fact two fetuses had plasma CA I11 levels above the 95th percentile and a third had a level on the 95th percentile. These findings were corroborated by concomitant elevations in CK levels.