Stephen P. Coburn

Alkaline Phosphatase Knock-Out Mice Recapitulate the Metabolic and Skeletal Defects of Infantile Hypophosphatasia†

Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock‐out mouse strains as potential models for hypophosphatasia. Homozygous mice (–/–) had < 1% of wild‐type plasma TNSALP activity; heterozygotes had the predicted mean of ∼50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′‐phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock‐out mice. Skeletal disease first appeared radiographically at ∼10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock‐out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.

Marrow Cell Transplantation for Infantile Hypophosphatasia

An 8‐month‐old girl who seemed certain to die from the infantile form of hypophosphatasia, an inborn error of metabolism characterized by deficient activity of the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP), underwent the first trial of bone marrow cell transplantation for this heritable type of rickets. After cytoreduction, she was given T‐cell‐depleted, haplo‐identical marrow from her healthy sister. Chimerism in peripheral blood and bone marrow became 100% donor. Three months later, she was clinically improved, with considerable healing of rickets and generalized skeletal remineralization. However, 6 months post‐transplantation, worsening skeletal disease recurred, with partial return of host hematopoiesis. At the age of 21 months, without additional chemotherapy or immunosuppressive treatment, she received a boost of donor marrow cells expanded ex vivo to enrich for stromal cells. Significant, prolonged clinical and radiographic improvement followed soon after. Nevertheless, biochemical features of hypophosphatasia have remained unchanged to date. Skeletal biopsy specimens were not performed. Now, at 6 years of age, she is intelligent and ambulatory but remains small. Among several hypotheses for our patients survival and progress, the most plausible seems to be the transient and long‐term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and perhaps chondrocytes, to ameliorate her skeletal disease.

A versatile cation-exchange procedure for measuring the seven major forms of vitamin B6 in biological samples

A cation-exchange procedure for detecting nanogram quantities of all seven common vitamin B6 metabolites in a variety of biological samples is presented. The procedure uses gradient elution with a single column to effect separation of the seven compounds within 40 to 60 min, depending on the complexity of the sample. Postcolumn addition of buffer and bisulfite ion permits sensitive fluorometric detection of all B6 compounds at the same wavelengths (330 nm activation, 400 nm emission).

Pyridoxine deficiency affects biomechanical properties of chick tibial bone

The mechanical integrity of bone is dependent on the bone matrix, which is believed to account for the plastic deformation of the tissue, and the mineral, which is believed to account for the elastic deformation. The validity of this model is shown in this study based on analysis of the bones of vitamin B6-deficient and vitamin B6-replete chick bones. In this model, when B6-deficient and control animals are compared, vitamin B6 deficiency has no effect on the mineral content or composition of cortical bone as measured by ash weight (63 +/- 6 vs. 58 +/- 3); mineral to matrix ratio of the FTIR spectra (4.2 +/- 0.6 vs. 4.5 +/- 0.2), line-broadening analyses of the X-ray diffraction 002 peak (beta 002 = 0.50 +/- 0.1 vs. 0.49 +/- 0.01), or other features of the infrared spectra. In contrast, collagen was significantly more extractable from vitamin B6-deficient chick bones (20 +/- 2% of total hydroxyproline extracted vs. 10 +/- 3% p < or = 0.001). The B6-deficient bones also contained an increased amount of the reducible cross-links DHLNL, dehydro-dihydroxylysinonorleucine, (1.03 +/- 0.07 vs. 0.84 +/- 0.13 p < or = 0.001); and a nonsignificant increase in HLNL, dehydro-hydroxylysinonorleucine, (0.51 +/- 0.03 vs. 0.43 +/- 0.03, p < or = 0.10). There were no significant changes in bone length, bone diameter, or area moment of inertia. In four-point bending, no significant changes in elastic modulus, stiffness, offset yield deflection, or fracture deflection were detected. However, fracture load in the B6-deficient animals was decreased from 203 +/- 35 MPa to 151 +/- 23 MPa, p < or = 0.01, and offset yield load was decreased from 165 +/- 9 MPa to 125 +/- 14 MPa, p < or = 0.05. Since earlier histomorphometric studies had demonstrated that the B6-deficient bones were osteopenic, these data suggest that although proper cortical bone mineralization occurred, the alterations of the collagen resulted in changes to bone mechanical performance.

Mild autosomal dominant hypophosphatasia: In utero presentation in two families

We describe four pregnancies in two families in which mild hypophosphatasia, apparently transmitted as an autosomal dominant trait, manifested in utero as severe long bone bowing. Postnatally, there was spontaneous improvement of the skeletal defects. Recognition of this presentation for hypophosphatasia by family investigation and assessment of the fetal skeleton for degree of ossification and chest size using ultrasonography is important. The prognosis for this condition is considerably better than for more severe forms of hypophosphatasia and for many other disorders that cause skeletal defects with long bone bowing in utero.

Location and turnover of vitamin B6 pools and vitamin B6 requirements of humans.

Our interest in vitamin B, was the result of a variety of evidence that vitamin B, metabolism was altered in Downs syndrome. At another New York Academy of Sciences conference in 1969, McCoy et al. summarized several studies suggesting that Downs syndrome was accompanied by either decreased size or increased turnover of the vitamin B, pool. Studies in our laboratory provided evidence of decreased pyridoxal phosphate concentrations in platelets2 and increased response of plasma concentrations to vitamin B, supplement^.^ When the United States Department of Agriculture (U.S.D.A.) Competitive Grant Program was announced in 1978, we decided to submit a proposal for further studies in Downs syndrome. However, as we attemped to describe the methods we would use, we realized that existing methods for assessing vitamin B, status would probably be inadequate for elucidating the mechanism responsible for any differences in vitamin B, metabolism between persons with Downs syndrome and normal controls. Therefore, just a few days before the proposal was due, the entire focus shifted from the original idea of studying vitamin B, metabolism in Downs syndrome to developing an entirely new method of assessing vitamin B, metabolism. Because Downs syndrome is caused by having an extra set of the genes carried on the 21st chromosome, the biochemical effect should be an increase in the rate of certain reactions. Changes in rate are most easily detected using isotope tracers. While some studies using radioactive tracers to examine vitamin B, metabolism had been conducted in h ~ m a n s , ~ we proposed using stable isotope tracers in order to eliminate the radiation hazard. When we asked a colleague where we might find the mass spectrometry resources to support such an undertaking, he suggested Dr. Peter Klein at Argonne National Laboratory. Dr. Klein agreed to assist us and has continued his interest since assuming his current position as Director of the Stable Isotope Laboratory at the U.S.D.A. Childrens Nutrition Research Center in Houston. As we discussed the project, Dr. Klein noted that because stable isotopes pose no hazard, rather than the single-bolus design frequently used with radioactive tracers, we could administer the isotopes over a long period of time. Adoption of that suggestion has been one of the key factors in the success of our work. However, first we had to synthesize the deuterated compounds, develop ways of analyzing the vitamers,* and develop the mass spectrometric techniques.

B6 vitamers: cation exchange HPLC☆

B 6 vitamers are extracted with trichloroacetic acid (from plasma, serum, erythrocyte, urine, or solid samples such as animal tissues) and quantitated using ternary gradient cation-exchange liquid chromatography with fluorescence detection. Fluorescence of pyridoxal and pyridoxal phosphate is enhanced through addition of a post-column buffer containing bisulfite.

Chapter 6 – Modeling Vitamin B6 Metabolism

This chapter discusses the modeling of vitamin B6 metabolism. The approaches to examining the kinetics of vitamin B6 metabolism are: (1) to examine individual enzymes or tissues; (2) to examine the changes with time after administration of unlabeled or labeled vitamin B-6 to intact animals or humans; (3) to gain insights into the metabolic state of vitamin B6 through the use of compartmental analysis. The models of vitamin B6 metabolism should allow for differences between oral and intravenous administration of label. A few tissues have the ability to conserve vitamin B6 during periods of low intake, making turnover dependent on intake. The conservation and flushing effects observed in vitamin B6 metabolism can be simulated by binding relationships. Monitoring isotope excretion in pyridoxic acid after simultaneous administration of D2-pyridoxamine, D3-pyridoxal, and D5-pyridoxine provides a practical way to examine short-term metabolism of these compounds.

A Critical Review of Minimal Vitamin B6 Requirements for Growth in Various Species with a Proposed Method of Calculation

We conclude that vitamin B6 is efficiently conserved by the tissues and that the major dietary requirement is to meet physiological stresses such as growth, pregnancy, lactation, vigorous exercise, and/or disease. A reasonable approximation of the minimal requirement for growth in most species can be obtained by assuming 15 nmol vitamin B6 for each g gain in body weight. For birds and fish, which can achieve 1 g gain/g feed, the vitamin B6 content would need to be about 15 mumol (3 mg)/kg feed. Such an intake is achievable with a variety of natural diets and would also be adequate for most mammalian species, which have lower feed efficiencies. Although well-balanced natural diets are usually adequate, supplementation with 5-10 mumol (1-2 mg)/kg diet is inexpensive and would provide a safety factor to allow for variations in feed composition and physiological stresses such as pregnancy, disease, parasites, and so on. At this time, whether the metabolic changes associated with increases in vitamin B6 intake above the minimum confer long-term health benefits on the organisms is not clear. Excessive vitamin B6 intake can be toxic. Fish and prawn seem to be particularly sensitive to this toxicity. Additional work is needed to clarify the interactions between vitamin B6 requirements and protein intake.

Separation of seven B6 vitamers by two-dimensional thin-layer chromatography.

Abstract Quantitative study of vitamin B 6 metabolism is complicated by the multiple forms in which B 6 may occur. The seven most common forms are: pyridoxine, pyridoxal, pyridoxamine, their 5′-phosphates, and pyridoxic acid. Contractor and Shane (1) have described a column chromatographic procedure for separating all except pyridoxine phosphate. Colombini and McCoy (2) reported an electrophoresis procedure which included all forms except pyridoxic acid. Several workers (3–5) have used thin-layer chromatography to separate various combinations of B 6 vitamers. However, we believe the technique described below is the first report of a simple method for separating all seven of the major B 6 compounds on a single thin-layer plate. It was developed from solvents suggested by Burg (6) and Sallay (personal communication) to follow the metabolism of labeled pyridoxine in tissues.