Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts.
BACKGROUND
In vitro, bone alkaline phosphatase (BALP) is released from the osteoblast membrane with its glycosylphosphatidylinositol (GPI) anchor still attached (i.e., in an anchor-intact form); however, in vivo, BALP circulates as a variable mixture of anchorless isoforms, which can be identified by high-performance liquid chromatography (HPLC). Previous studies have shown that the relative abundance of these BALP isoforms in serum may be clinically useful for the diagnosis and management of metabolic bone disease.


METHODS
In the current studies, we describe a method for the determination of anchorless BALP isoforms in extracts of bone and we present novel data on the conversion of anchor-intact to anchorless BALP by incubation with endogenous circulating GPI-specific phospholipase D (GPI-PLD).


RESULTS
A 72-h extraction with 0.1% Triton X-100 released approximately 90% of the BALP activity from powdered bone. An average of 19% of this activity was anchorless, but essentially all of the activity could be converted to the anchorless form by incubation with partially purified GPI-PLD from human serum. Using HPLC, we detected four BALP isoforms (B/I, B1x, B1, and B2) in these GPI-PLD-treated extracts of bone. An additional BALP fraction was also detected in the samples during the initial phase of GPI-PLD treatment.


CONCLUSIONS
The abundance of the BALP isoforms differed between bone and serum, particularly for the B/I isoform, which comprised, on average, 18% of the BALP in GPI-PLD-treated extracts of healthy bone tissue, but only 6% of the total BALP activity in serum from healthy individuals. Based on our recent finding of differences in the number of sialic acid residues between the BALP isoforms, we hypothesize that this difference between BALP isoforms in serum and extracts of bone is due to the different patterns of glycosylation, which results in different biological half-lives in the circulation. A preliminary application of our method to the extraction of BALP isoforms from a small number of human bone samples suggests that the method should be useful for studies of human skeletal site-specific and metabolic bone disease-specific differences in the amounts and distributions of the BALP isoforms in bone.
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000469:Alkaline Phosphatase / Q000097:blood / Q000378:metabolism*; D001842:Bone and Bones / Q000201:enzymology*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005269:Femur / Q000201:enzymology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D012516:Osteosarcoma / Q000201:enzymology / Q000473:pathology; D010739:Phospholipase D / Q000378:metabolism; D014018:Tissue Distribution
D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D000469:Alkaline Phosphatase; D010739:Phospholipase D; C065017:glycoprotein phospholipase D