Heat Inactivation in the Study of Human Alkaline Phosphatases

Posen, Solomon; Neale, F. C.; Clubb, John S.

doi:10.7326/0003-4819-62-6-1234

Cited by 180 publications

(42 citation statements)

References 15 publications

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“…Serum alkaline phosphatase activity rose in patients 1 and 2 beginning about 8 weeks after vitamin D, treatment was stopped. Over 80% of the augmented activity was heat labile, indicating bone as its major source (33). Alkaline phosphatase activity in serum plateaued at a level two-to threefold above that present during maintenance therapy with vitamin D,, Radiographic examinations of long bones and skull revealed mild progressive rachitic changes emerging after withdrawal of antirachitic treatment.…”

Section: Analysis Of Datamentioning

confidence: 96%

Response to Crystalline 1α-Hydroxyvitamin D3 in Vitamin D Dependency

Glorieux

et al. 1975

Pediatr Res

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ExtractThe therapeutic response to chemically_synthesized la-hydroxycholecalciferol (la-OH-D,) was studied in three patients with autosomal recessive vitamin D dependency (ARVDD). The daily maintenance dose for vitamin D,, to prevent signs of vitamin D deficiency in these patients, was 40-54.5 fig/kg, or about 100 times normal (Table 1). Withdrawal of maintenance therapy with vitamin D, resulted in the ultimate reappearance of the vitamin D depletion syndrome in patients I and 2 ( Figs. 1 and 2). The third patient presented with the deficiency syndrome despite adequate vitamin D nutrition and was recognized to have ARVDD.Treatment with la-OH-D, by mouth in all three patients at dose levels of 1-3 pg/24 hr (8@100 ng/kg) corrected hypocalcemia and suppressed parathyroid hormone-dependent renal loss of amino acids (Figs. 1,2, and 4). Rickets healed in 7-9 weeks on la-OH-D, alone (Fig. 3 ) The therapeutic response was rapid. It was usually seen first in the rise of serum calcium (Figs. 5 and 6). Withdrawal of la-OH-D, was followed first by a fall of serum phosphorus, then by a fall in serum calcium; the latter occurred within about 2 weeks of withdrawal.Because the synthesis of la-OH-D, is simpler than for la,25-dihydroxycholecalciferol and because the former is an effective therapeutic analog of vitamin D hormone, we believe these studies in ARVDD reveal la-OH-D, to be the agent of choice for treatment of this and analogous diseases. SpeculationVitamin D dependency or pseudodeficiency rickets is believed to be an inborn error of vitamin D hormone biosynthesis. The putative abnormal enzyme is 25-hydroxycholecalciferol 1-hydroxylase in the recessively inherited trait. Consequently, this experiment of nature offers a special opportunity to examine the requirement in human subjects, for la-hydroxyvitamin D, metabolites.Patients with ARVDD (14, 18, 36) develop signs of severe postnatal vitamin D deficiency, despite a nutritional intake of vitamin D, or vitamin D, (40) that would prevent rickets in normal subjects; hence the term pseudodeficiency preferred by some investigators (34). Persistent hypocalcemia appearing soon after birth is accompanied by an excess of circulating parathyroid hormone (I) which in turn is associated with hyperphosphaturia and hyperaminoaciduria (1, 37). Elevated serum alkaline phosphatase activity, severe rachitic bone lesions, and enamel hypoplasia affecting teeth that form postnatally complete the clinical syndrome ( I , l I, 14, 18, 34, 36). Maintenance treatment with vitamin D, only at levels about 100 times the normal requirement fully reverses the manifestations of deficiency hence the term vitamin D dependency (18,36).The origin of the disturbed physiology in ARVDD lies in a selective disturbance of calcium absorption by intestine (18). A defect either in the biosynthesis of the active hormone form of vitamin D or in the ability of target organ(s) to respond to vitamin D hormone has been proposed (36) to explain the dependency on vitamin D,, D,, or 25-OH-D, of patients with ARVDD. ARVDD...

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Section: Analysis Of Datamentioning

confidence: 96%

Response to Crystalline 1α-Hydroxyvitamin D3 in Vitamin D Dependency

Glorieux

et al. 1975

Pediatr Res

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“…Reduction in activity is clearly demonstrated in the case of bone ALP, and inhibition ofisoenzymes running to a similar position can be inferred from the experiments on yGT. This could be due in the case of bone ALP to heat inactivation, liver and intestinal isoenzymes being relatively stable (Posen, 1965). On the other hand, evidence from thislaboratory (M. Sullivan, unpublished) indicates thatyGT is stable after heating for 30minutes at 56°C.…”

Section: Meanmentioning

confidence: 94%

Quantitative Recovery of Alkaline Phosphatase and γ-Glutamyl Transferase Isoenzymes after Polyacrylamide Gel Electrophoresis

Brooks¹,

Sammons²

1980

Ann Clin Biochem

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SUMMARY Loss of activity of the isoenzymes of v-glutamyl transferase and alkaline phosphatase has been shown to occur during electrophoresis on polyacrylamide gel. After studying the possible factors concerned in this loss, reasonable recovery from the gel can be obtained only for the isoenzyme staying at the origin. Maximum recovery is 60% for origin y-glutamyl transferase and 92 % for origin alkaline phosphatase.Recent work from this department indicates that although the isoenzymes of y-glutamyl transferase (yGT) are well separated when using polyacrylamide gel electrophoresis, and easily identified by staining, the actual recovery is only 30 % of the original activity. As this is a most effective and clinically useful medium for separating not only the isoenzymes of yGT (Azzopardi and Jayle, 1973) but also those of alkaline phosphatase (ALP) (Walker and Pollard, 1971;Price and Sammons, 1974), it seemed desirable to investigate this phenomenon further, firstly, to see if ALP behaved similarly, secondly, to see if the extent of recovery was similar for all the isoenzymes separated, and, thirdly, to identify if possible the factors involved. Methods ELECTROPHORESISShandon disc electrophoresis equipment was used for both enzymes. The tubes were 7·5 ern in length with a bore of 0·5 cm. Gels were prepared for yGT by the method of Azzopardi and Jayle (1973) and for ALP by the method of Smith et al. (1968). These use respectively 7 % and 5 % polyacrylamide made up in Tris-glycine buffer pH 8'3 and Tris-borate buffer pH 9·5for optimum separation of the isoenzyrnes, A 20~l serum sample was applied to the gel after dilution 1:1 with 40 %sucrose. The mobility of the separation was marked by pre-staining the diluted serum with a grain of bromophenol blue. Separation was carried out at 1·0 rnA/gel constant current for 5 minutes to allow for penetration into the gel and then raised to "Present address: Group Pathology Laboratory, Lakin Road, Warwick. 154 rnA/gel until the albumin-bromophenol blue complex reached the bottom of the gel. After electrophoresis the gels were rimmed from the tubes and the enzymes were measured in the appropriate length of gel. Each 0·5 ern had a volume of 0'1 ml and contained 35 mg (for yGT) and 25 mg (for ALP) of polyacrylamide. ENZYME METHODS v-Glutamyl transferaseA manual adaptation of the kinetic yGT assay of Rosalkiand Tarlow (1974) using y-L-glutamylp-nitraniJide as substrate at a pH of 8'0 and at 37°C was used. The reaction was stopped by adding 25 %acetic acid. The change in optical density at 410 nm was then used as a measure of activity. The coefficient of variation of the method was found to be 7·2 %.Alkaline phosphatase A manual modification of the optimised conditions recommended by the Committee on Enzymes (1977) using p-nitrophenyl phosphate was used. After incubation for 30 minutes, the reaction was stopped by the addition of 0·4M NaOH. The optical density was read at 410 nm. Controls were assayed by adding 0'4M NaOH before the addition of substrate. The coefficient of variatio...

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“…It is practicable in the presence of an increased liver band, and may be useful as an index of active bone repair, for example, in assessing the union of fractures. Electrophoresis is as simple as, and more specific than, separation of the isoenzyme by heat (Posen, Neale, and Clubb, 1965). Many like to measure a separate enzyme whose serum activity increases in cholestasis but not in bone disease, eg, 5' nucleotidase or y-glutamyltranspeptidase, the latter being more sensitive but less specific for disease of the liver (Whitfield, Pounder, Neale, and Moss, 1972).…”

Section: Methodsmentioning

confidence: 99%

Plasma alkaline phosphatase isoenzymes in hepatobiliary disease

Afonja

Baron

1974

J Clin Pathol

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By cellulose acetate or acrylamide gel electrophoresis it is possible to separate these alkaline phosphatase isoenzymes from serum: [anode] fast liver, slow liver, placenta/Regan, bone, intestine, bile [cathode]. Heat or chemical inhibition can confirm the differentiation.Normal adult serum always contains slow-liver isoenzyme, and sometimes bone isoenzyme: the latter is always present in serum of children. In hepatobiliary disease slow-liver isoenzyme was always increased: intestinal isoenzyme appeared in many cases of cirrhosis (of blood groups B and 0) but fast-liver and bile isoenzymes were occasionally seen in miscellaneous cases. The findings in other diseases included Regan isoenzyme in six out of 45 cases of malignant disease.

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Heat Inactivation in the Study of Human Alkaline Phosphatases

Cited by 180 publications

References 15 publications

Response to Crystalline 1α-Hydroxyvitamin D3 in Vitamin D Dependency

Response to Crystalline 1α-Hydroxyvitamin D3 in Vitamin D Dependency

Quantitative Recovery of Alkaline Phosphatase and γ-Glutamyl Transferase Isoenzymes after Polyacrylamide Gel Electrophoresis

Plasma alkaline phosphatase isoenzymes in hepatobiliary disease

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