A high resolution PIXE measurement for blood plasma ultrafiltrate. Application to loosely bound copper.

A new technique for investigation of elemental concentrations in subfractions of blood plasma is presented. The method is composed of the ultrafiltration of plasma in the presence of ethylenediaminetetraacetic acid (EDTA) and the measurement of the elemental composition by proton-induced X-ray emission (PIXE). The blood samples were collected from both healthy persons and patients suffering from breast cancer. The main emphasis in this study was on the determination of loosely bound copper (Cu) in plasma subfractions containing substances with molecular mass under 10,000, but zinc (Zn) and iron (Fe) contents of these fractions were also determined. The detection limits obtained with this method for Cu, An and Fe were approximately 10 ppb (wet wt).

Early breast cancer, diet, and plasma copper fractions.

In a study of diet and early breast cancer, blood plasma copper has been analyzed by Proton Induced X-ray Emission analysis as both total copper (P-Cu) and that ultrafiltrable from plasma (P-edu-Cu) through membranes with a cut-off at molecular mass 10,000 after equilibration with disodium ethylene diamine tetraacetic acid (EDTA) at 4 degrees C. Ceruloplasmin (P-cer) was also measured using nephelometry of anticeruloplasmin monoclonal antibody-ceruloplasmin complexes. Dietary copper intake per day (D-Cu) was assessed over a five-day dietary record period and calculated from dietary components using a computer program. P-edu-Cu correlated significantly with both D-Cu and ceruloplasmin while P-Cu correlated only with ceruloplasmin. Further, ceruloplasmin did not significantly correlate to D-Cu. Hence, P-edu-Cu better reflects copper status than do P-cer or P-Cu as it relates to both the major copper enzyme in plasma and to daily copper intake. This may be important in drawing conclusions about the significance of copper in disease states where copper fractions other than ceruloplasmin may be most important owing, for example, to oxidative properties. Categorization as cancer or normal, by copper parameters (D-Cu, P-edu-Cu, P-Cu, P-cer), was studied in multiple correlation. In particular, the ratio P-cer/P-Cu and the ratio P-edu-Cu/D-Cu were significantly related to disease. Irrespective of age (pre- and post-menopausal), highly significant differences between normals and early stage breast cancer patients were seen with p < 0.0001 to p < 0.01. The precise role played by plasma and dietary copper fractions deserves continued attention in view of the present and earlier results in cancer studies.

Evidence for increased non-ceruloplasmin copper in early-stage human breast cancer serum.

We measured total serum copper and ceruloplasmin levels in pre- and postmenopausal Stage I and II breast cancer (BC) patients and omnivorous and vegetarian controls. The omnivorous groups included 14 premenopausal women [33 +/- 6 (SD) yrs] and 11 postmenopausal women (57 +/- 5 yrs), and the vegetarian groups were comprised of 12 premenopausal subjects (34 +/- 7 yrs) and 11 postmenopausal subjects (59 +/- 5 yrs). There were 13 premenopausal BC patients (39 +/- 7 yrs) and 10 postmenopausal BC patients (66 +/- 6 yrs). Fasting serum samples were taken on three consecutive days, typically four times in the year. Serum ceruloplasmin levels (g/l) were measured by nephelometry utilizing monoclonal antiserum, and total serum copper levels (mumol/l) were determined by proton-induced X-ray emission analysis. Premenopausal patients had higher serum copper levels than their controls (mean 18.7 vs. 16.6, p less than 0.03). For ceruloplasmin, the postmenopausal BC patients had significantly lower levels than pooled postmenopausal controls (0.309 vs. 0.370, p less than 0.001). The copper-to-ceruloplasmin ratio was significantly higher in the pooled cancer groups than in the pooled control groups (3.69 vs. 3.21, p less than 0.001), with similar patterns in both pre- and postmenopausal classes. This high serum copper-to-ceruloplasmin ratio in BC patients may reflect disordered copper metabolism in this disease, which could also have implications for the origin of, or the response to, the cancer process.

Trace element studies on whole human cerebrospinal fluid with external beam PIXE.

External beam PIXE analysis with a 2.4 MeV proton beam was used to determine the concentrations of K, Ca, Fe, Cu, Zn and Br in cerebrospinal fluid from patients having various disorders. The obtained total concentration ranges K 34,000-1,079,000, Ca 5300-81,300, Fe 40-1030, Cu 20-1650, Zn 15-1250 and Br 400-43,000 micrograms/kg are compared with the values given in the literature. In certain patients there were very high CSF bromine levels, but this was shown to be the result of taking medications presented as bromide salts. The possibility of using the method in clinical practice for CSF analysis is considered. The new method of preparing self-supporting films of the samples was used. This method was further optimized by investigating in detail the use of EDTA as a homogenizer.

A new rapid method for quantification of nitrogen in human serum employing the 14N(p,p' gamma)14N reaction: application to human pregnancy.

The total nitrogen concentrations in dried serum from 54 pregnant women and 17 newborn babies were determined by a new application of the 14N(p,p' gamma)14N reaction resonance at 3.9 MeV. The samples were bombarded in a He atmosphere by 4.1 MeV protons from a tandem Van de Graaff accelerator. The mean dry-weight nitrogen concentration in serum sampled during early pregnancy (6-12 weeks; 13.9 g per 100 g +/- 5.4%) was significantly higher than that in serum sampled during late pregnancy (38-42 weeks: 13.0 g per 100 g +/- 3.9%, p less than 0.001) and than that in serum taken from the umbilical cord (13.3 g per 100 g +/- 4.6%, p less than 0.01). The nitrogen levels measured using this rapid nuclear technique, applied for the first time to human serum analysis, agree well with parallel Kjeldahl analyses.

Whole body cellular and collagen nitrogen in healthy and wasted man.

The classical literature shows that wasting of body protein takes place mainly from the cellular compartment leaving high proportions of extracellular collagen. Whole body nitrogen ( WBN ) is proportional to whole body protein (cellular + extracellular) while whole body potassium ( WBK ) is almost entirely intracellular. WBK might be an adequate index of cellular wasting. WBN was measured by neutron activation and WBK by counting 40K in 29 healthy males and 131 male and female patients with wide ranges of body composition. Our wasted patients with Crohn's disease or ulcerative colitis, had higher WBN / WBK ratios than matched healthy controls and the difference between the two groups was in the cellular ratio (23.6 mol of N/mol of K, 0.33 g of N/mmol of K, 3 mmol of K/g of N). A multiple regression model for all the subjects represented cellular nitrogen by a term in WBK and extracellular nitrogen by simple anthropometric measurements. The partial regression coefficient of WBN on WBK was 22.6 +/- 1.1 (SE) mol of N/mol of K; this was also similar to the cellular ratio. Our results were compatible with extracellular protein (mainly collagen, which is 35% of normal whole body protein) remaining resistant to wasting even when severe loss of cellular protein occurs. The high ratios of WBN / WBK in wasted patients can be explained by this disproportionate wasting of cellular substances and they do not imply alterations in the cellular N/K ratio. We suggested that the stable ratio of WBK to cellular protein makes it an effective index of cellular wasting. The resistance of collagen to wasting and the preponderance of extracellular mass in the fat-free mass of wasted subjects, make WBN and fat-free mass unreliable guides to the extent of wasting.

Selenium and other elements in human maternal and umbilical serum, as determined simultaneously by proton-induced X-ray emission.

Using PIXE (proton-induced X-ray emission), we simultaneously determined the concentrations of Se, Ca, Fe, Cu, Zn, Br, and Pb in blood serum from 56 pregnant women, 25 healthy controls, and 31 others with twin pregnancy or some complicating condition (diabetes, hypertension, epilepsy, hepatosis gravidarum, pre-eclampsia, small baby), and in cord-blood serum from 21 newborns. Pellets, pressed from the serum samples after addition of yttrium as an internal standard, mixing, and evaporating at 30 degrees C with or without reduced pressure (less than 1 kPa), were bombarded by 2.2 MeV protons from a Van de Graaff accelerator in the air and the induced X-rays collected by a Ge(Li) detector. Relative to mean Se values for early six- to 12-week pregnancy (0.045 ppm), those for 35-42 week pregnancy (0.028 ppm) were low (p less than 0.001). Umbilical cord blood serum showed even lower values (0.016 ppm, p less than 0.001)--findings in harmony with the incidence pattern of Keshan cardiomyopathy. Pb crossed the placenta; values for cord serum were not significantly different from those in pregnancy serum. Cu, Zn, Fe, and Ca showed the significant expected patterns in the different groups. Compared with the late-pregnancy controls, Fe was high in mothers of small-birth-weight babies (1.70 ppm, p less than 0.02). Br was high in pre-eclampsia (3.59 ppm, p less than 0.05) and mothers with twins (3.61 ppm, p less than 0.05).

Application of the law of mass action to design and performance of receptor assays for 1,25(OH)2 vitamin D.

The binding of 1,25(OH)2 vitamin D3 to its chick duodenal cytosol receptor protein has been analysed on a law of mass action model. Within the limits of experimental error there is such good agreement, when only one binding site is considered, that it is possible to fit the standard assay curve for practical use. Simple pocket-calculator methods are given for this. The equilibrium constant for the reaction, under the conditions used (pH 7.4, 25 degrees C), was 2.1 X 10(10) 1 X mol-1. A kinetic analysis under the same conditions yielded forward and backward reaction rate constants of 6.5 X 10(6) mol-1 X s-1 X l(2) and 3.1 X 10(-4) s-1 X 1, respectively. The kinetic curve also very closely fitted to the experimental data on the basis of the model. The theory for use of the mass action model to optimize the binding-site concentration to give a minimum absolute error for any measured ligand concentration is presented. The use of the model in assay design, standard curve fitting, quality control and problem solving are discussed.

Assay for plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 by "high-performance" liquid chromatography.

We describe a new, rapid, and simple column-chromatographic procedure for 25-OH vitamin D3 in plasma. The vitamin is extracted by use of Sep-pak C18 (octadecyl alkylated silicic acid), a short factory-packed reversed-phase column, and 25-OH vitamin D2 + 25-OH vitamin D3 fraction is eluted with methanol/water. The 25-OH D2 and 25-OH D3 are then well resolved on a high-resolution 3-micrometer silicic acid straight-phase liquid-chromatography column. The peaks are quantified against a 25-OH D3 standard by ultraviolet absorbance. Recovery was assessed by use of tritiated 25-OH D3. The within-assay coefficient of variation of the method was 5% and recovery 93%. The method was evaluated with 26 samples from control subjects and 17 samples from patients, seven with liver disease and 10 who had undergone ileo-ileostomy for hypercholesterolemia. The normal seasonal variation was observed for 25-OH D3 concentrations, and they correlated negatively and significantly with those of 25-OH D2. Post-ileo-ileostomy concentrations of 25-OH D3 in plasma were generally similar to those in normal individuals for the same reason (winter), but 25-OH D2 concentrations were insignificantly lower. The patients with chronic liver disease had significantly lower 25-OH D3 concentrations than normal persons but higher 25-OH D2 concentrations, with a significantly higher 25-OH D2/25-OH D3 ratio, indicating poor storage of vitamin D3.

A sensitive and simplified receptor assay for 1,25(OH)2 vitamin D.

A sensitive receptor assay for 1,25(OH)2 vitamin D using 2.5-5 ml plasma and a small incubation volume (less than 150 microliters) is described. The standard curve is plotted as free/total counts vs in-assay ligand concentration and is fitted to a law of mass action model. The conditions in the assay are: pH 7.4(0.05 mol/l phosphate buffer), 11% v/v ethanol concentration, and 1 g/l total protein concentration. The reaction of the receptor protein and ligand is satisfactorily complete after 2.5 h at 25 degrees C. The bound fraction is precipitated by 40% polyethylene glycol in ethanolic phosphate buffer, separated by centrifugation, and the supernatant drawn off and counted. The radioactivity used to trace the binding reaction is part of that originally added to the plasma. There were no interferences in the assay from vitamin D3, 25(OH) vitamin D3 or 24,25(OH)2 vitamin D3. After further purification by high pressure liquid chromatography there was no reduction in the 1,25(OH)2 vitamin D values measured directly after LH-20 chromatography. The plasma levels in 20 normal men and 19 women were 106 +/- 36 pmol/l (mean +/- SD) and 105 +/- 40 pmol/l, respectively. These results compare well with those reported from other centres. The intra-assay coefficient of variation in the useful range of measurement is about 11%.

Plasma profile of hydroxylated vitamin D metabolites: methods and results in normals for spring-winter in Southern Finland.

The spring-winter plasma concentrations of the hydroxylated metabolites of vitamin D have been measured in normal human subjects (20 male, 13 female), using high pressure liquid chromatography with UV-detection for 25-hydroxy vitamin D3/D2 (25(OH)D) and 24,25-dihydroxy vitamin D3/D2 (24,25(OH)2D) and a radioligand receptor assay for 1,25-dihydroxy vitamin D3/D2 (1,25-(OH)2D). The respective levels were 26.6 +/- 16 nmol . l-1, 2.24 +/- 1.3 nmol . l-1 and 114 +/- 35 pmol . l-1. These are in good agreement with levels from other centres for the same season. There was a significant negative correlation between 24,25(OH)2D and 1,25(OH)2 levels. The possible significance of this in the context of the low post-winter body burden of vitamin D is discussed. There was a positive correlation between 25(OH)D and 24,25(OH)2D levels but it did not reach statistical significance. There were no statistically significant differences between the levels of the hydroxylated metabolites in men and women but the 24,25(OH)2D levels were generally lower in the women. The 1,25(OH)2D levels were similar to those measured by isotope dilution mass fragmentography on Norwegian donor blood.

In vitro measurement of phosphorus in small bone biopsies using 14 MeV neutron activation analysis.

In studying diseases of bone such as osteoporosis and renal osteodystrophy it is important to have an index of change in bone mineral content. For this purpose we describe a method for measuring the phosphorus content of 100--150 mg bone biopsies. Femoral and iliac crest biopsies from 6 cadavers were each paired with a standard bone of similar size and irradiated with 14 MeV neutrons for 10 minutes. A special rotor ensured the same activating flux across both sample and standard bone which were both wrapped in aluminium foil. The 28Al generated from bone phosphorus (31P(n,alpha)28Al) and the 27Mg generated from the aluminium foils (27Al(n,p)27Mg), which undergo beta-decay have useful gamma rays at 1779 keV and 844 keV respectively. These gamma rays were counted using a 110 cm3 Ge(Li) detector and a rotating frame permitting four 30 s counts of sample and standard alternately (interval for rotation 10 seconds) in identical geometries. Using this system the phosphorus content of the biopsies could be determined with a coefficient of variation of 4.6% using the standard bone as reference and 6% using aluminium foil as reference. In dense bone the P/dry weight ratio was 30--50% greater than in spongy bone. The clinical value of the method is discussed.

Elemental analysis of bone mineral by backscattering of alpha particles.

Backscattering of 2 MeV alpha particles has been applied for the first time to studies of the inorganic substances of human bone. Bone samples taken from the femoral shaft (dense bone) and iliac crest (spongy bone) of 18 cadavers were analysed for the calcium, phosphorus and oxygen content, after lyophilisation and heating for 1.5 h at 500 degrees C to remove water and organic material. The backscattering method produces a spectrum in which all the elements present in the sample except hydrogen can be seen at once. The reproducibility of the measurements was less than 2%. No previous studies were found in the literature of the measurement of elemental oxygen in bone samples. Ca/P ratios varied from 1.97 to 2.47, Ca/O ratios from 0.76 to 0.91 and P/O ratios from 0.34 to 0.41, the latter two being about 10--15% lower than expected if bone mineral were hydroxyapatite (Ca/O = 0.96 and P/O = 0.44). The results are consistent with previous work on Ca/P ratios but the low Ca/O and P/O ratios could not be explained without postulation of crystal or hydroxyl water in bone mineral. Support for the explanation was gained by differential gravimetric and thermal analysis.

In vivo measurement of calcium by the 37Ar method: a study of the effect of recirculating breath collection systems on the exhalation rate.

A study has been made of the effect that different methods of breath collection have on the measurement of calcium in vivo by the 40Ca(n,alpha) 37Ar method. The effect of open and rebreathing breath collection methods has been examined by using intravenous injections of 133Xe. This study has indicated that comparison of calcium measurements between individuals and between centres is likely to be accurate only if breath is collected on 'open circuit'.

Problems in the interpretation of the in vivo measurement of calcium by the argon-37 method: an investigation of inert-gas elimination in humans.

An investigation has been made of some physiological problems associated with the interpretation of in-vivo measurements of calcium by the argon-37 method. Inert-gas elimination in humans over a period of several days was studied using i.v. injections of Xe-133. The results imply that the exhalation rate of A-37 formed in bone will be affected by the individual's composition, in particular body fat. Comparison of calcium measurements between individuals and between laboratories is meaningful only if corrections are made for differing individual composition.

Measurement of whole body calcium in chronic renal failure: effects of 1alpha-hydroxyvitamin D3 and parathyroidectomy.

In vivo neutron activation analysis was used to measure changes in whole body calcium in patients with chronic renal failure. Eight patients, not treated by haemodialysis, showed losses of calcium in contrast to increases noted in twenty-one patients treated by haemodialysis. Changes in whole body calcium were independent of pre-existing renal bone disease. Treatment of bone disease by total parathyroidectomy (six patients) or 1alpha-hydroxyvitamin D3 (eleven patients) resulted in large increases in total body calcium. In patients given 1alpha-hydroxyvitamin D3, changes in total body calcium correlated with histological and radiological indices of response but variable changes in forearm bone mineral content were observed. Neutron activation analysis provides a convenient method for measuring skeletal mass and may also be useful in the assessment of response to 1alpha-hydroxyvitamin D3 of patients with osteitis fibrosa and osteomalacia.

Spinal calcium changes with 1alpha-hydroxyvitamin D3.

During treatment of renal osteodystrophy with 1alpha-hydroxyvitamin D3 in eleven patients, regional changes in the skeleton have been compared with long-term calcium balance as assessed by whole-body calcium. Radial bone changes did not correlated well with calcium balance, but spinal calcium changes were of a similar magnitude when changes were large. Bone alkaline phosphatase changes correlated well with changes in spinal calcium, but less well with changes in radial bone density.