Solomon Posen

THE MECHANISM OF THE ELEVATION OF SERUM ALKALINE PHOSPHATASE IN PREGNANCY

SERUM alkaline phosphatase is elevated in late pregnancy. This phenomenon was first described 30 years ago (Coryn, 1934) and has since been repeatedly confirmed (Cayla and Fabre, I935 ; Meranze et al., 1937; Young et al., 1946). The reason for this increase in enzyme activity has been the subject of considerable speculation. Earlier theories included increased osteoblastic activity in the mother (Ramsay et al., 1938) and the passage of foetal osteoblastic enzyme into the maternal circulation (Kerleau and Cayla, 1939; Ebbs and Scott, 1?40), while a number of more recent workers have suggested a placental origin of the additional alkaline phosphatase (Jung and Stark, 1956; Klees and Frenzel, 1960; Kubli, 1961). Beck and Clark (1950) attempted to distinguish placental from other alkaline phosphatases by means of taurocholate inhibition and concluded that the increase in serum enzyme during pregnancy was due to taurocholate resistant (i.e., placental) material. Unfortunately, resistance to bile salt inactivation is not unique to placental enzyme but is shared by intestinal alkaline phosphatase and by over half the alkaline phosphatase in non-pregnant sera. Boyer (1961) showed, by means of starch gel electrophoresis, that fractions of the alkaline phosphatase from the sera of pregnant women migrate in the same zones as alkaline phosphatase from human placentae. However, electrophoretic separation of iso-enzymes on starch gel is not suitable for the routine laboratory, nor does this method lend itself readily to quantitative estimations. Neale et al. (1964a, b) described a simple method to discriminate between human alkaline phosphatase of placental and non-placental origins. Unlike alkaline phosphatase from other human sources (Moss and King, 1962) the placental enzyme is unaffected by heating at 56” C . for 30 minutes. By heating serum to this temperature, one is able to measure the concentration of heatstable enzyme which remains. This paper reports our studies to determine the presence or absence of heat-stable alkaline phosphatase in sera of normal individuals, women in various stages of pregnancy and newborn infants. The heat stability of alkaline phosphatase from various female reproductive organs has also been determined.

The action of EDTA on human alkaline phosphatases

Abstract The effects of EDTA on the human alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) of bone, intestine and placenta, have been studied by means of automated methods employing controlled concentration gradients. EDTA has three effects of these phosphatases: (a) In the presence of excess substrate, low concentrations of EDTA (10−5-10−3 M) cause a loss of phosphatase activity which is the same irrespective of the EDTA concentration. (b) Above 10−3 M EDTA bone and intestinal phosphatases display an increasing loss in activity with increasing concentrations of EDTA. Placental phosphatase, however, displays a progressive gain in activity with increasing concentration of EDTA. (c) Preincubation of the phosphatases with EDTA results in a time-dependent inactivation which is not reversed by dilution. This inactivation is also temperature-dependent and pH dependent. Alkaline phosphatases from different tissues show different susceptibilities to the irreversible inactivation by EDTA. The controlled concentration gradients were also used to study the kinetics of the action of EDTA on these phosphatases. Complex kinetics were observed with all alkaline phosphatases.

Action of urea on human alkaline phosphatases: with a description of some automated techniques for the study of enzyme kinetics

Abstract An automated method is described for the study of enzyme kinetics. It employs a controlled substrate concentration gradient so that reaction velocities can be studied at a large number of substrate concentrations. Urea has two effects on human alkaline phosphatases: a reversible instantaneous inhibition and an irreversible time-dependent inactivation. The reversible inhibition is of the noncompetitive type. Alkaline phosphatases from different tissues show different susceptibilities to irreversible inactivation by urea. The placental enzyme is the most resistant, while bone enzyme is the most sensitive of the alkaline phosphatases examined. The alkaline phosphatases in human sera resemble those of the presumptive tissues of origin in their susceptibility to the actions of urea.

The origin of serum alkaline phosphatase in the rat

Abstract The increase in alkaline-phosphatase activity which occurs in the serum of the rat after feeding is due, in part, to material inactivated by anti-rat intestinal-alkaline-phosphatase antibody. Feeding also causes the rats serum alkaline phosphatase to resemble rat intestinal-alkaline-phosphatase in its relative heat resistance, in its electrophoretic migration pattern on starch gel and in its l -phenylalanine sensitivity. Rat intestinal alkaline phosphatase injected intravenously into rats disappers from the circulation of the recipient animals within 2 h. Qualitative differences are described between the alkaline phosphatase obtained from the rats upper small intestine and that obtained from the lower small intestine.

PLACENTAL ALKALINE PHOSPHATASE AND PREGNANCY

Human placental alkaline phosphataset resembles other mammalian alkaline phosphatases by its close association with the surface membrane of epithelial cells. The maternal surfaces of the syncytiotrophoblastic cells are especially rich in alkaline phosphatase,3 and there is reason to believe that placental microvilli project into the maternal uterine sinusoids in much the same fashion as intestinal microvilli project into the intestinal lumen. Some particulate trophoblastic material is shed into the maternal circulation4 in the same way as particulate matter from the intestinal wall is desquamated into the gut lumen.5 The structure of human placental alkaline phosphatase was studied by Harkness, who concluded that human placental alkaline phosphatase has a molecular weight of approximately 1 25,000,s and that, like other alkaline phosphatases, it is a zinc metalloenzyme. As yet, no physiological function has been established for placental, or indeed for any other, mammalian alkaline phosphatase. The assumption that this group of enzymes is somehow connected with active transport rests on their location within the cell, on their ability to bind inorganic pho~phate ,~ and on evidence obtained with bacterial alkaline phosphatases.s Placental alkaline phosphatase injected into human subjects has no measurable effect on the metabolism of the recipient^,^ and L-phenylalanine a known inhibitor of intestinal alkaline phosphatase is without effect on the absorption of glucose or inorganic phosphate from the everted gut loop.10 Placental alkaline phosphatase shares with nonplacental alkaline phosphatases the ability to hydrolyze a large number of substrates,11st2 including inorganic pyrophosphate13 and 4-methylumbelliferyl ph0~phate.l~ Harkness.* who determined the rates of hydrolysis of a number of substrates at the same substrate concentration in the same buffer, at the same buffer strength, and at the same pH found that, mole for mole, slightly more beta-glycerophosphate than paranitrophenylphosphate was hydrolyzed per unit of time by placental alkaline phosphatase. Human placental alkaline phosphatase differs from nonplacental alkaline phosphatases15 by its remarkable resistance to heat denaturation (TABLE 1 ) . Some of this heat stability is lost during butanol extraction, acetone precipitation and dialysis (TABLE 2) but even crystalline human placental alkaline phosphatase has been shown to be relatively heat resistant.l2 The heat resistance of human placental alkaline phosphatase appears to be species-specific. All nonhuman placental alkaline phosphatases examined so far have been denatured to varying degrees at 56 C. (TABLE 3). Heat stability is accompanied by a resistance to denaturation during preincubation with u ~ e a . ~ ~ J Human placental alkaline phosphatase may be pre-

Human salivary alkaline phosphatase

Abstract Human salivary secretions were tested for the presence of alkaline phosphatase. The activity was greatest in mixed saliva and least in parotid saliva. Dialysis increased alkaline phosphatase in all types of salivary secretions, particulary in parotid saliva. This effect is attributed to the presence in high concentrations of inorganic phosphate in salivary secretions. Patients with elevated serum alkaline phosphatase have normal salivary alkaline phosphatase.

Parathyroid and Calcium Disorders

Publisher Summary This chapter describes parathyroid and calcium disorders. In primary hyperparathyroidism, there is hyperplasia or neoplasia of parathyroid tissue with hypersecretion of parathyroid hormone. As a rule, only one of the parathyroid glands is involved and in the large majority of cases, hyperparathyroidism occurs as an isolated abnormality. In a few hyperparathyroid patients, particularly those suffering from other endocrine disorders, such as acromegaly, hypergastrinemia, and familial hyperparathyroidism, all parathyroid tissue may be involved in the hyperplastic process. Parathyroid carcinomas are rare. Excessive activity by the parathyroid glands results in high blood levels of parathyroid hormone (PTH) and in various biochemical abnormalities secondary to the effects of PTH. There is enhanced renal excretion of inorganic phosphate with hypophosphatemia. Bone resorption is enhanced resulting in hypercalcemia and hypercalciuria. The associated increase in osteoblastic activity may lead to elevation in serum alkaline phosphatase. PTH stimulates the synthesis of cyclic AMP and 1,25-dihydroxyvitamin D by renal tubules so that urinary cyclic AMP and serum 1,25-dihydroxyvitamin D are increased.

45 – Calcium Homeostasis

Publisher Summary nThis chapter describes tests focusing on calcium homeostasis. It discusses the assay of nephrogenous cyclic AMP and the protocol involved. After an overnight fast (water may be taken ad libitum), the patient drinks 200 ml of water at 7 A.M. and a further 200 ml at 7.30 A.M. At 8 A.M., the patient empties his bladder and discards the urine. The patient then collects all urine voided during the next 2 hr. During this period, the patient drinks as much water as possible to ensure an adequate urinary volume (some protocols call for 200 ml every 30 min). Blood (15 ml) is taken into an ethylenediaminetetraacetic acid tube in ice at 9 A.M. A final urine specimen is voided at 10 A.M. The 2-h urine volume is noted and an aliquot is kept for cyclic AMP and creatinine assays. The blood is centrifuged as soon as possible after collection and the plasma kept at −20°C until assay. The chapter also presents an overview of the steroid suppression test and parathyroid hormone infusion.