Cold lability of lactate dehydrogenase isoenzymes and the effective preparation of reference material for clinical laboratory use.

To evaluate the storage of samples and enzyme reference materials, and to improve the commutability for inter-laboratory surveillance of activity values of lactate dehydrogenase (LD; EC 1.1.1.27) in clinical laboratory medicine and in animal veterinary medicine, we studied the electrophoretic patterns and cold lability of LD isoenzymes from tissue sources of some common vertebrate species and also from human serum sources. Among many isoenzymes from these sources, only rat LD fractions showed similar electrophoretic patterns to those of human sera, and rat LD-1 fraction was relatively cold-stable. Total LD and isoenzyme LD-1 activities in routine laboratory samples and quality-control sera were measured using eight kinds of commercially available LD assay kit, including lactate and pyruvate substrate systems. Coefficients of variation between these assay kits were markedly reduced when the activities were calculated using the partially purified rat LD-1 fraction as an enzyme reference material, compared with the activities calculated using the factor indicated in each assay kit. In the regression analysis, the intercept and slope were calculated for the regression equations obtained from 12 pairs of these assay kits. The values obtained from a small amount of human serum and control serum samples were within the 95% confidence regions of those from larger amounts of human serum samples by using the present rat LD-1 standard for measuring LD-1 activities with lactate substrate. It was evident that a cold-stable and homogeneous LD isoenzyme as an enzyme reference material might contribute to accurate measurement of activities in heterogeneous samples for inter-laboratory quality-assurance surveys in both human and animal clinical laboratory use.

Phylogenic study of denaturation of lactate dehydrogenase isoenzymes from different species by high and low temperature.

We studied the influence of storage at different temperatures on lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes from different tissues and different species, and analysed biochemical and biophysical mechanisms of denaturation during storage. Isoenzymes obtained from tissue extracts of mammals, poultry, reptiles. amphibians and fish were shown to have their own denaturation ranges at low temperatures by post-treatment assays and transition temperature analysis. These ranges were between -10 and -20 degrees C for most vertebrate LD isoenzymes. Circular dichroism analysis indicated that the denaturation of LD isoenzymes was probably caused by a change in the hydrophobic interactions in the molecule. At higher temperatures, LD-1 isoenzyme was more thermostable than LD-5 from the same animal species, except for rats, the LD-5 activity of which was more thermostable than the LD-1 activity. These findings indicate that variable effects of storage of samples and reference materials at low temperatures should be considered, and that it is necessary to establish LD isoenzyme standards for animal clinical laboratory investigations.

4-(5',6'-Dimethoxybenzothiazolyl)benzoyl fluoride and 2-(5',6'-dimethoxybenzothiazolyl)benzenesulfonyl chloride as sensitive fluorescence derivatization reagents for amines in high-performance liquid chromatography.

4-(5',6'-Dimethoxybenzothiazolyl)benzoyl fluoride (BHBT-COF) and 2-(5',6'-dimethoxybenzothiazolyl)-benzenesulfonyl chloride (BHBT-SOCI) have been developed as highly sensitive and selective fluorescence derivatization reagents for primary and secondary aliphatic amines in HPLC. These reactivities were investigated using n-propylamine, n-heptylamine and N-methylhexylamine as model compounds to optimize the derivatization conditions. Both reagents readily reacted with the amines in basic media to give the corresponding fluorescent derivatives, which were separated isocratically by reversed-phase (C18) liquid chromatography with aqueous methanol. The detection limits (signal-to-noise ratio = 3) of BHBT-COF for primary and secondary amines are approximately 3 and 30 fmol, respectively, and those of BHBT-SOCI are approximately 3 and 300 fmol, respectively, for an injection volume of 20 microliters. Both reagents gave no fluorescent derivatives for aromatic amines.

Immunohistochemically detectable duct-like structures in benign and malignant eccrine poromas: CEA and involucrin immunostaining.

The duct-like structures present in 7 cases of benign and malignant eccrine poroma were examined by immunohistochemical staining for carcinoembryonic antigen (CEA), involucrin, and S-100 protein. The demonstration of CEA and involucrin was helpful in the recognition of these structures. The overt immunopositivity precedes morphological evidence for duct formation. On the basis of the CEA immunostaining, the duct-like structures were divided into 4 types: 1) mature acrosyringeal structure, 2) cystic luminal structure lined by elongated cells, 3) immature acrosyringeal structure, and 4) vacuole- or dot-like potential lumen in a single cell. Involucrine was observed in the lining cells of 1) and 2). None of the 4 types showed positive reactivity for S-100 protein, suggesting the irrelevance of these structures to the secretory element of sweat gland. The polymorphism of the ductal formation tended to be more remarkably observed in malignant eccrine poromas than in the cases of benign eccrine poroma and poroepithelioma tested.

Erythematosquamous skin lesions in hereditary lactate dehydrogenase M-subunit deficiency.

Peculiar erythematosquamous lesions were observed in two adult patients in Japan with hereditary lactate dehydrogenase M-subunit deficiency. Although these patients showed excessive fatigue and myoglobulinuria after extended exercise, they were usually asymptomatic. However, nonpruritic follicular papules or erythematous patches with scaly edges were present on the extensor surfaces of the extremities of these patients since childhood, showing some improvement after puberty. There were also erythematous patches on the weight-bearing areas of their soles. These patches showed exacerbation and a tendency toward peripheral spreading in summer. These skin lesions provide an important clue in the detection of this genetic enzyme deficiency.

Interrelationship between water-barrier and reservoir functions of pathologic stratum corneum.

Scaly skin lesions are caused by decreased water content of the stratum corneum, despite the well-known fact that they usually show increased water passage. By performing simultaneous measurements of transepidermal water loss (TEWL) to check the water-barrier function of the stratum corneum and cutaneous conductance to the high-frequency electric current of 3.5 MHz, which is an indicator of skin-surface hydration state, in patients with psoriasis who had lesions of various grades of severity, we obtained data indicating that there is an inverse relationship between these functions of the stratum corneum. Furthermore, a time course study of TEWL and functional analysis of stratum corneum by an in vivo water sorption-desorption test performed on the experimentally induced scaly lesions after adhesive-tape stripping demonstrated that such pathologic stratum corneum is characterized by a water-holding defect that is associated with increased TEWL.

Texture of the skin graft, with special reference to the hydration state of the stratum corneum.

The water contained in the superficial portion of the stratum corneum plays an important role in keeping the skin surface soft and smooth. In an attempt to study the true nature of the "texture" of grafted skin, in vivo water sorption-desorption tests were performed on grafts, on normal skin adjacent to grafts, and on normal skin surrounding the donor sites. The results showed that skin grafted to sites other than the face maintained a functional similarity to normal skin surrounding the donor site. However, in skin grafted to the face, the functions of the stratum corneum, as measured in terms of hydration state, increased, and this was associated with an improved water-holding capacity that probably results from the effect of abundant sebum and sweat excretion in adjacent skin. From these results it is suggested that the recipient site plays an important role in determining the difference in the function of the stratum corneum, i.e., the "texture" between grafts on the face and those on other sites.

Water sorption-desorption test of the skin in vivo for functional assessment of the stratum corneum.

Based on the evidence from our previous studies that we can evaluate the hydration state of the skin surface quickly and quantitatively in terms of conductance to the high frequency electric current of 3.5 MHz, we have established a simple in vivo function test that furnishes information on the hygroscopic property and water-holding capacity of the stratum corneum in a few minutes. The test procedure consists of electromeasurements before and after application of a droplet of water on the skin for 10 seconds to obtain data on the hygroscopic property of the skin surface and later serial measurements at an interval of 30 seconds for 2 min to evaluate the water-holding capacity. Under usual ambient conditions normal skin surface showed a high rise in conductance just after application of water, which was followed by a rapid fall-off within 30 seconds, thereafter by gradual return to the prehydration levels by 2 min. By this method we have demonstrated that (i) the superficial horny layer of normal skin is much less hygroscopic and less capable of holding water than the corresponding deeper portions and that (ii) scaly skin shows functional defects in both hygroscopicity and water-holding capacity, between which the former normalizes much faster than the latter.