Glenville Jones

Metabolic Bone Disease in Patients Receiving Long-Term Total Parenteral Nutrition

We have prospectively investigated calcium and bone metabolism in 16 patients receiving total parenteral nutrition for periods ranging from 7 to 89 months. In 12 patients, bone biopsies at 6 to 73 months after the start of parenteral nutrition showed osteomalacia. Plasma 25-hydroxyvitamin D levels were normal in all patients. Seven persons developed hypercalcemia, and 10 had hypercalciuria with a negative calcium balance. Serum phosphorus was normal and plasma parathyroid hormone level, normal or decreased. Three patients with the severest form of the disease had vitamin D withdrawn from their solutions. Subsequently, urinary calcium decreased, and serum calcium became normal; two persons reverted to a positive calcium balance. Thus, patients receiving total parenteral nutrition may develop metabolic bone disease characterized by osteomalacia, hypercalcemia, hypercalciuria, and a negative calcium balance. This may be caused by both defective mineralization and increased bone resorption induced by vitamin D, its metabolites, or another unrecognized factor.

A Possible Role of Vitamin D in the Genesis of Parenteral-Nutrition-Induced Metabolic Bone Disease

Patients receiving long term parenteral nutrition may develop metabolic bone disease. In all 11 patients studied, histologic studies of bone showed excessive unmineralized bone tissue despite normal plasma 25-hydroxyvitamin D levels. Three patients also had bone pain and fractures and severe urinary loss of calcium and phosphate. Withdrawal of vitamin D from parenteral nutrition solutions was associated with improved histologic findings of bone in all patients, shown by a decrease in osteoid tissue and an increase in tetracycline uptake. In the three patients with symptoms, bone pain subsided, fractures healed, and urinary loss of calcium and phosphate decreased. Thus, vitamin D may be a factor in the genesis of parenteral nutrition-induced metabolic bone disease.

Comparative determination of plasma cholesterol and triacylglycerol levels by automated gas--liquid chromatographic and autoanalyzer methods.

Plasma samples obtained during a prevalence study of hyperlipemia in a free living urban population were analyzed for total cholesterol and triacylglycerol content by automated high-temperature gas--liquid chromatographic (GLC) and automated colorimetric (Auto-Analyzer, AA11) methods. The analyses were done over a three-year period. The methods gave excellent overall correlation for both total cholesterol (r = 0.9811) and total triacylglycerols (r = 0.9739). Detailed comparisons of the results obtained by the two methods with natural samples over the entire concentration range, indicated that the GLC method gave cholesterol values 5--10 mg% lower and triacylglycerol values 10--20 mg% lower than the corresponding AA11 determinations. The differences between the two methods are attributed to an overestimation of the cholesterol and triacylglycerol levels by the AA11 method due to presence of variable amounts of interfering chromogens in the plasma extracts. The between-method relative error ranged from 3 to 5% for cholesterol and from 5 to 10% for triacylglycerols. The within-day standard deviation of GLC averaged 2.3 mg% for cholesterol and 3.5 mg% for triacylglycerols. The between-day standard deviation of the GLC method averaged about 6 mg% for both cholesterol and triacylglycerols. The within-day, within GLC, relative error averaged 1.12% for cholesterol and 2.66% for triacylglycerols. The apparent high precision and high accuracy of the GLC method recommend it as an alternative to the indirect methods of plasma cholesterol and triacylglycerol analysis, especially where a smaller throughput of samples is not a limitation and where both total amount and composition of the lipids is of interest.

Lipid class and molecular species interrelationships among plasma lipoproteins of normolipemic subjects

As evidence of lipoprotein interconversion and/or equilibration, a gas-liquid chromatographic (GLC) examination was made of the lipid class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of seven male and four female normolipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by GLC of the intact cholesterol and glycerol esters and of ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and an increasing phospholipid and cholesteryl ester content when progressing from the very low (VLDL), to the low (LDL2) and high (HDL) density lipoproteins, as already established by conventional analyses. However, the LDL2 contained about a two times higher proportion of total phospholipids as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher molecular weight species than the sphingomyelins of either VLDL or LDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding fractions of lipoproteins. In general, the lipid class and molecular species distribution is incompatible with the hypothesis which postulates VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin:cholesterol acyltransferase. The significant differences noted in the lipid class and molecular species distribution suggest that the true transformations of the lipoproteins are much more complex and may also involve cholesteryl ester-triacylglycerol, triacylglycerol and phosphatidylcholine exchanges via appropriate carrier plasma proteins, as well as possible phase separation of lipids during the removal of the excess surface material from the VLDL remnants, as already demonstrated in in vitro experiments. It is concluded that a direct GLC analysis of the neutral and polar lipid components of plasma lipoprotein classes provides important evidence of lipoprotein interrelationships which may be utilized to test existing and new hypotheses of lipoprotein interconversion.

Comparative determination of plasma phospholipids by automated gas—liquid chromatographic and manual colorimetric phosphorus methods

Plasma samples obtained during a prevalence study of hyperlipemia in a free-living urban population were analyzed for phosphatidylcholine, sphingomyelin and lysophosphatidylcholine content by automated high-temperature gas--liquid chromatographic (GLC) and manual colorimetric phosphorus (thin-layer chromatographic, TLC) methods. The GLC estimates were obtained from a quantitative analysis of the diacylglycerol, ceramide and monoacylglycerol moieties released from the parent phospholipids by digestion with phospholipase C, while the TLC estimates were derived by manual colorimetric phosphorus analyses of the individual phospholipid classes resolved by TLC. On samples analyzed over a two-year period the methods gave excellent correlation for the total phospholipids (r = 0.98), phosphatidylcholine (r = 0.98) and sphingomyelin (r = 0.90), but resulted in a poor agreement for lysophosphatidylcholine (r = 0.69). Comparable results were obtained for estimates of these phospholipids in plasma very low density, low density and high density lipoproteins. The between-method coefficient of variation ranged from 3 to 5% for phosphatidylcholine and from 5 to 10% for sphingomyelin. The relative error for the estimates of lysophosphatidylcholine ranged from 10 to 25%, and was due to the inclusion in the GLC estimates of a variable proportion of plasma free monoacylglycerols. Other differences between the two methods are due to various analytical errors and biases inherent in the two techniques. The within-day, within GLC, relative error averaged 1% for phosphatidylcholine, 3% for sphingomyelin and 5% for lysophosphatidylcholine. The apparent high precision and accuracy of the GLC method recommend it as an alternative to conventional direct methods of phospholipid analyses based on TLC isolation of lipid classes and colorimetric measurements of their phosphorus content. The GLC analyses of the plasma phospholipids are particularly convenient in conjunction with GLC measurements of plasma cholesterol and triacylglycerols, where a smaller throughput of samples is not a limitation and where both total amount and relative proportion of the lipids are of interest.

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography.

Abstract This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)2D3, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)2D and 1,25-(OH)2D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D3 metabolites from their vitamin D2 analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)2D3 and 24(R),25-(OH)2D2 as well as 1,25-(OH)2D3 and 1,25-(OH)2D2.

Pathogenesis of rickets in chronic hepatobiliary disease in children

To investigate whether hepatobiliary rickets is caused by defective intestinal absorption of vitamin D or by impaired hepatic hydroxylation of the vitamin, we studied three children who developed severe rickets, hypocalcemia, and hypophosphatemia, two despite having received 400 to 800 IU vitamin D per day by mouth, and one despite prolonged treatment with 10,000 IU daily. On oral vitamin D therapy, plasma vitamin D and 25-hydroxyvitamin D levels were low. When two children were treated with weekly intravenous doses of 3,000 IU vitamin D to approximate the recommended prophylactic allowance, their plasma calcium and phosphate values improved promptly, the radiographic lesions healed, and the plasma concentrations of vitamin D and 25-hydroxyvitamin D became normal. Our studies indicate that the primary cause of hepatobiliary rickets is intestinal malabsorption of vitamin D, not impairment of the hepatic metabolism of the vitamin.

Chromatographic separation of24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone using a cyanobonded phase packing

Abstract A high-performance liquid chromatographic system is described for the baseline resolution of 25-hydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone, the three principal circulating metabolites of vitamin D3 in the vitamin D-replete animal. The system is based upon a cyano-bonded phase packing and the solvent hexane—isopropanol—methanol (94:5:1). Of particular interest is the strong retention of carbonyl-containing vitamin D metabolites. The new system can be used for unequivocal analysis of vitamin D metabolites in plasma samples from clinical and animal studies and in the separation and identification of renal metabolites generated in vitro.

1534 EFFECT OF CHRONIC METABOLIC ACIDOSIS ON THE RENAL METABOLISM OF 25-OH-D3 IN THE PERFUSED KIDNEY

Chronic acidosis has been shown to influence the renal metabolism of 25-OH-D3 in the intact rat. In this study we asked the question whether the pH of the animal directly influences activities of 1- and 24-hydroxylases. Acidosis was induced in vit.D deplete [D(−)] and vit.D replete rats [D(+)] by feeding 3% NH4Cl in drinking water for 9 days. Using an isolated perfused kidney system, we compared 1- and 24-hydroxylase activities (in fmole product/hr/g, tissue) in kidneys from D(−) and D(+) rats respectively. Results are Mean ± SEM.Acidosis significantly inhibited 1-hydroxylase but not 24-hydroxylase activity. This change in 1-hydroxylase activity correlated directly with blood pH (r=0.79, p<0,001). Although blood ionic Ca was higher in D(−) acidotic rats (0.83±0.03 mM) compared with controls (0.58±0.04 mM), 1-hydroxylase activity did not correlate significantly with blood ionic Ca (r=0.48, 0.10>p> 0.05) . We conclude that an increase in [H]+ concentration directly suppress 1-hydroxylase activity but does not suppress 24-hydroxylase activity.

PROTEIN SYNTHESIS INHIBITORS INHIBIT 24, 25-DIHYDROXY-VITAMIN D3 SYNTHESIS IN THE D-DEPLETED KIDNEY

The final step of activation of vitamin D occurs in the kidney Depending upon the circulating levels of Ca, Pi and D, 24,25-and 1,25-dihydroxyvitamin D are produced in varying amounts by renal mitochondrial hydroxylases. 24,25-(OH)2D3, the major metabolite present under physiological conditions, may have a role in bone formation.We have studied the biochemical control of 24,25-(OH)2D3 synthesis. When kidneys isolated from D-replete rats were perfused, they produced mainly 24,25-(OH)2D3. Kidneys from D-depleted animals produced 1,25-(OH)2D3. However, they began to synthesize 24,25-(OH)2D3 2 to 3 hours after starting perfusion when the substrate was 10 ng/ml 25-hydroxyvitamin D3 (25-OHD3). We investigated whether the conversion of 3H-25-OHD3 to 3H-24,25(OH)2D3 was dependent upon the synthesis of new protein. We added the protein synthesis inhibitor actinomycin D (4×10−6M) or cycloheximide (1×10−5M) to the perfusate. Addition of the inhibitors between 0 and 3 hours of perfusion abolished conversion of 3H-25-OHD3 to 3H-24,25(OH)2D3 (control 2.76 ± 0.31 % vs inhibitor 0.30 ± 0.20%). However, when they were added 3 or 4 hours after starting perfusion, the synthesis of D metabolite was not inhibited (control 2.76 ± 0.31% vs inhibitor 3.95 ± 0.6%). The results suggest that the initiation of 24,25-(OH)2D3 production requires the synthesis of new protein.