25-Hydroxyvitamin D and vitamin D in human milk: effects of supplementation and season.

Breast-milk 25-hydroxyvitamin D (25-[OH]D) and vitamin D were measured in mothers supplemented with 2000 or 1000 IU (50 or 25 micrograms) of vitamin D/d or with no supplementation. Fore- and hindmilk samples were collected at two stages of lactation (8 and 15 or 20 wk after delivery) and at different seasons. Season affected the levels of 25-(OH)D and vitamin D. The 25-(OH)D levels were higher in hind- than in foremilk. Supplementation had no effect on vitamin D levels. Milk 25-(OH)D levels of mothers receiving either 1000 or 2000 IU (25 or 50 micrograms) vitamin D/d were significantly higher than those of unsupplemented mothers in February and April. In theory, supplementation with 2000 IU (50 micrograms) vitamin D should have increased the calculated antirachitic activity of the milk in winter to the levels of unsupplemented mothers in September; however, responses varied widely among individuals.

Bone mineral metabolism after total gastrectomy.

Gastric surgery is mostly needed for treatment of gastric malignancy. To investigate the effect of total gastrectomy on bone mineral density (BMD) and bone mineral metabolism we evaluated 18 patients after total gastrectomy. Mean interval since operation was 71 +/- 20 months. BMD results were compared with age- and gender-matched controls (n = 46) and also expressed as T and Z scores. Bone mineral density measured by dual-energy X-ray absorptiometry (DXA) was found to be significantly lower in patients after total gastrectomy compared with healthy controls in the lumbar spine (p = 0.017 for women, p = 0.002 for men), femoral neck (p = 0.004 for women, p = 0.001 for men), Ward's triangle (p = 0.031 for women, p = 0.003 for men), and greater trochanter (p = 0.001 for women, p = 0.001 for men). Z scores for lumbar spine, femoral neck, Ward's triangle, and greater trochanter were -0.83, -1.54, -1.02, and -1.19, respectively. Biochemical measurements correlated poorly with BMD and were found to be of lesser value in diagnosing reduced bone mass as well as in differential diagnosis of etiology of osteopenia. The results of our study show the deleterious effect of total gastrectomy on bone mineral status and suggest an increased fracture risk in these patients.

A high-stearic acid diet does not impair glucose tolerance and insulin sensitivity in healthy women.

Results in epidemiological and experimental studies suggest that a diet rich in saturated fat may affect insulin sensitivity. However, no published data are available on the effect of stearic acid in this respect. Therefore, we examined the effects of a high-stearic acid diet and a high-oleic acid diet on glucose metabolism, serum lipids and lipoproteins, and blood coagulation factors in 15 healthy female subjects. Subjects followed the two experimental diets for 4 weeks according to a randomized crossover design. Both experimental diet periods were preceded by consumption of a baseline diet for 2 weeks. The diets provided 36% of energy (E%) as fat. In the experimental diets, 5 E% stearic or oleic acid was substituted for 5 E% of saturated fatty acids in the baseline diet. After the experimental diets, no differences were found in the insulin sensitivity index (mean+/-SEM, 5.4+/-1.9 v 5.2+/-1.6 x 10(-4) min(-1) x microU(-1) x mL(-1), nonsignificant [NS]), glucose effectiveness (0.026+/-0.006 v 0.026+/-0.003 min(-1), NS), or first-phase insulin reaction ([FPIR] 368+/-57 v 374+/-66 mU/L x min, NS). The concentration of serum lipids and lipoproteins and blood coagulation factors did not differ after the diet periods. In conclusion, a diet rich in stearic acid did not deteriorate glucose tolerance or insulin action in young healthy female subjects as compared with a diet rich in oleic acid.

Urinary bone resorption markers in monitoring treatment of symptomatic osteoporosis.

We have studied the clinical usefulness of urinary bone resorption markers in postmenopausal women with symptomatic osteoporosis. The study design is a randomised double-blind placebo controlled study, in which the subjects were daily treated for 24 months either with a hormone analogue (2.5 mg Livial, generic name Tibolone, Organon, Amsterdam, Holland) plus 800 mg calcium (n = 14, age 63+/-5 years, range 52-68 years), or with placebo plus 800 mg calcium (n = 19, age 66+/-7 years, range 50-75 years). The laboratory methods for urinary bone resorption markers were enzyme immunoassays (EIA) for urinary pyridoline (PYD) and deoxypyridoline crosslinks (DPD), and for cross-linked N-telopeptides of Type I Collagen (NTx), and an HPLC assay for urinary hydroxyproline (HOP). All the urine assay results were calculated per mmol creatinine. All the resorption markers decreased during the two-year study period in both groups. The Z scores (discriminating power, i.e. ability of the different tests to distinguish the hormone treated subjects from the placebo treated subjects) for HOP and PYD were rather low: 0.06-1.52 for HOP and 0.68-1.47 for PYD. The differences between the two treatment groups were statistically significant for DPD at 12 and 24 months of treatment (P = 0.0471 and P = 0.0466, respectively), the Z scores ranging 0.45-1.90. NTx showed the most prominent decrease from the beginning of the study especially in the hormone treatment group: the differences between the two treatment groups were statistically highly significant for NTx already at 6 months of treatment (P = 0.0015), and the Z scores remained high ranging 2.11-3.82 throughout the two-year study period. Dual X-ray absorptiometry (DXA) of the lumbar spine and femoral neck did not show statistically significant differences between the two treatment groups throughout the two-year study period. After 2 years there was, however, a significant increase in bone density both in the spine (+ 6.6%, P = 0.0002) and in the femoral neck (+ 3.4%, P = 0.0389) in the women with hormone treatment. In the control group a significant increase (+ 5.1%, P = 0.0012) in the spine, whereas a non-significant decrease (-1.5%, n.s.) in the femoral neck was observed. We suggest that measurement of urinary cross-linked peptides derived from Type I collagen (NTx and DPD) might be a useful biochemical method of observing the positive clinical effect (i.e. reduction in bone resorption) following hormone replacement therapy in postmenopausal fracture patients.

An improved method for routine determination of vitamin D and its hydroxylated metabolites in serum from children and adults.

A method for routine determination of vitamin D and its major metabolites 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxy-vitamin D (1,25(OH)2D) in serum samples from normal children and adults has been developed. Methodological improvements enable a rapid and accurate analysis of 25(OH)D and also the microscale screening of other metabolites present in large concentrations in serum. Vitamin D and its metabolites are extracted from serum samples using hexane/propan-2-ol, which allows a convenient separation of the water soluble and lipid soluble fractions from each other and also from proteins. Preparative silicic acid chromatography was used to separate vitamin D from its metabolites and also from the major portion of co-eluting lipid contaminants. An automated LC solvent delivery and sample introduction system was used to achieve the rapid separation of metabolites. Vitamin D was further purified using adsorption high-performance liquid chromatography and assayed using reverse phase high-performance liquid chromatography connected with UV detection. The 25(OH)D fraction from the preparative chromatography was measured by a competitive protein-binding assay along with 24,25(OH)2D, which was purified by reverse phase high-performance liquid chromatography along with 1,25(OH)2D. A diluted human serum from a pregnant woman (3rd trimester of pregnancy) was used as source of the binding protein for 25(OH)D and 24,25(OH)2D. 1,25(OH)2D was determined by a competitive protein-binding assay using a diluted cytoplasmic 1,25(OH)2D receptor protein isolated from the intestinal mucosa of rachitic chicks. Vitamin D and its metabolite levels were assayed in serum samples from normal children and adults.

Does apolipoprotein E genotype relate to BMD and bone markers in postmenopausal women?

OBJECTIVES: Bone mineral density (BMD) and development of osteoporosis are partly determined by genetic factors. The associations between one of suggested candidate, apolipoprotein E (apo E) genotype to bone mineral density (BMD) and bone biochemical markers was studied in 464 subjects recruited from a population-based group of early postmenopausal women (n = 13100). Additionally, the influence of apo E genotype on BMD changes during a 5-year follow-up with or without hormone replacement therapy (HRT) was investigated. METHODS: Participants were randomized into two treatment groups: HRT group: Sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate with or without vitamin D3, 100-300 IU/day + calcium lactate, 500 mg/day (n = 232), and the non-HRT group: Calcium lactate, 500 mg/day alone or in combination with vitamin D3, 100-300 IU/day (n = 232). BMD was measured from the lumbar spine and proximal femur at baseline and after 5 years of treatment (n = 352). In a subgroup (n = 59), the serum concentrations of bone biochemical markers (intact osteocalcin (OC), bone-specific alkaline phosphatase (BAP) and type I collagen carboxy-terminal telopeptide (ICTP)) were measured at baseline and after 1 year of follow-up. RESULTS: At baseline, the BMDs were similar between the five apo E genotype groups (2/3, 2/4, 3/3, 3/4, 4/4). No significant differences in lumbar or femoral neck BMDs of women with the apo E4 allele were found compared with those without it. There was a statistically significant difference in 5-year BMD changes between the HRT and non-HRT groups. After 5 years, the BMD of the femoral neck had remained constant and the mean lumbar spine BMD had increased by 1.5% in the HRT group, whereas both BMDs had decreased by 4-5% in the non-HRT group. However, the apo E genotype did not modify the changes in BMD in either group. Additionally, the baseline concentrations of bone metabolic markers and their 1-year changes showed no genotype-related associations. CONCLUSIONS: The results of our population-based study indicate that apo E genotype does not modify lumbar or femoral neck BMDs or serum bone biochemical markers or their response to HRT in early postmenopausal Caucasian women.

Nephrotoxicity of tobramycin. Value of examining various protein and enzyme markers.

The value of various protein and enzyme markers for the assessment of early nephrotoxicity of tobramycin was studied in 18 patients with febrile infection. The renal clearance of creatinine and the excretion of the following protein and enzyme markers were measured on the first and the last day of the 5-8 day treatment period: albumin, N-acetyl-beta-D-glucosaminidase (NAG), beta 2-microglobulin, alpha-amylase, lysozyme and retinol-binding protein (RBP). Diurnal excretions of beta 2-microglobulin, lysozyme and RBP, all markers of tubular dysfunction, were already increased on the first treatment day, compared to similar control patients treated with non-aminoglycoside antibiotics and reference values of our laboratory. The excretion of NAG, an enzyme released from tubular cell lysosomes, was not increased initially, but on the last treatment day it was increased most consistently of all the markers studied. Glomerular filtration rate was halved in 5 of the patients. The results suggest that the initial increase in beta 2-microglobulin, lysozyme and RBP excretion is a result of an early tubular transfer block by tobramycin, whereas the late increase in NAG excretion probably reflects the progress of tubular cell damage during the course of aminoglycoside therapy.

Epidemiology of vitamins A, E, D and C in rural villages in Finland: biochemical, nutritional and socioeconomical aspects.

We carried out an extensive health profile analysis in spring-winter 1986 in four Eastern Finnish rural villages as a part of the Healthy Village Study. Altogether, 793 people at working age (20-64 years of age, 427 men and 366 women) participated (80%). Serum lipids (total cholesterol, HDL-cholesterol and triglycerides) and plasma vitamins (vitamin A, D, E and C) were measured as biochemical indicators of health. The dietary habits were reflected in high serum total cholesterol, and in low plasma vitamin C (ascorbic acid, mean 34.4 mumol/l in men, and 51.2 mumol/l in women). The plasma levels of the other vitamins studied were, in general, satisfactory. The mean plasma concentration of vitamin A (retinol) was 2.70 mumol/l in men, and 2.23 mumol/l in women. The gender, high body weight and the use of animal fats had the strongest association to apparent plasma retinol concentrations. The corresponding plasma concentrations of vitamin D (25-hydroxy-D) were 34.1 nmol/l and 35.4 nmol/l, and vitamin E (d-alpha-tocopherol) 22.1 mumol/l and 22.2 mumol/l. Vitamin D deficiency (plasma 25-OHD less than 12.5 nmol/l) was seen in 5% of the subjects. A good vitamin D status was correlated with the use of vitamin supplements, and, surprisingly, with the frequent consumption of alcohol.

Vitamin C deficiency and risk of myocardial infarction: prospective population study of men from eastern Finland.

OBJECTIVE: To examine the association between plasma vitamin C concentrations and the risk of acute myocardial infarction. DESIGN: Prospective population study. SETTING: Eastern Finland. SUBJECTS: 1605 randomly selected men aged 42, 48, 54, or 60 who did not have either symptomatic coronary heart disease or ischaemia on exercise testing at entry to the Kuopio ischaemic heart disease risk factor study in between 1984 and 1989. MAIN OUTCOME MEASURES: Number of acute myocardial infarctions; fasting plasma vitamin C concentrations at baseline. RESULTS: 70 of the men had a fatal or non-fatal myocardial infarction between March 1984 and December 1992.91 men had vitamin C deficiency (plasma ascorbate < 11.4 mumol/l, or 2.0 mg/l), of whom 12 (13.2%) had a myocardial infarction; 1514 men were not deficient in vitamin C, of whom 58 (3.8%) had a myocardial infarction. In a Cox proportional hazards model adjusted for age, year of examination, and season of the year examined (August to October v rest of the year) men who had vitamin C deficiency had a relative risk of acute myocardial infarction of 3.5 (95% confidence interval 1.8 to 6.7, P = 0.0002) compared with those who were not deficient. In another model adjusted additionally for the strongest risk factors for myocardial infarction and for dietary intakes of tea fibre, carotene, and saturated fats men with a plasma ascorbate concentration < 11.4 mumol/l had a relative risk of 2.5 (1.3 to 5.2, P = 0.0095) compared with men with higher plasma vitamin C concentrations. CONCLUSIONS: Vitamin C deficiency, as assessed by low plasma ascorbate concentration, is a risk factor for coronary heart disease.

A modification of the acid diazo coupling method (Malloy-Evelyn) for the determination of serum total bilirubin.

A simple and reliable method for the determination of total bilirubin from human serum is described. In this method, indirect bilirubin is liberated by the tenside in 0.12 mol l-1 HCl (R1), and the total bilirubin is coupled with a 2,5-dichlorobenzene diazonium (DBD) salt to obtain the corresponding azobilirubin having a lambda max of about 520-522 nm. The method can easily be applied to the KONE Delta, a fully automated, discrete random access clinical analyser, and also to less modern instruments. A sample volume of 5 microliters, R1 volume of 180 microliters, and R2 volume of 36 microliters was used on the KONE Delta. After a 5-min incubation at 37 degrees C, measurement at 575 nm was done (main wavelength). The within-run imprecision (CV%) varied from 2.9 to 0.3% within the serum total bilirubin range of 14-290 mumol l (n = 10). The between-run imprecision was from 2.2 to 1.3% within the range 13-97 mumol l-1 (n = 8). The method is linear up to at least 340 mumol l-1 (19.8 mg dl-1), and dilution extends the test limit to 3400 mumol l-1 (198.8 mg dl-1). The linearity of dilution was good over the practical measuring range. The present method had a strong linear correlation with the Boehringer 2,5-dichlorophenyl diazonium (DPD) method on the Hitachi 717 analyser: y(DBD) = 1.018x(DPD)+0.758, r = 0.9955 (n = 61). The stability of R2 (diazo reagent) in the analyser reagent compartment lasts at least 2 weeks.

Vitamin A, D and E status in a Finnish population--a multivitamin study.

The serum concentrations of the three fat-soluble vitamins A, D and E, and the serum transport proteins for the first of these, were measured during the winter and summer in a healthy adult Finnish population. No statistically significant differences were found in the levels of vitamins A and E between the seasons. However, a marked seasonal variation was found in the levels of vitamin D metabolites, the serum concentrations of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D being higher in summer, and that of 1,25-dihydroxyvitamin D somewhat higher in winter, whereas the concentration of vitamin D-binding globulin remained constant throughout. The serum concentrations of vitamins A and E showed a marked interrelationship with a significant correlation (r = 0.512, P less than 0.001, n = 80). No outright deficiencies of the vitamins were found in this study, but the serum concentrations of 25-hydroxyvitamin D were rather low in winter. Supplementation with a moderate dose of vitamins A and E over a 2-month winter period did not affect the serum concentrations of these vitamins, whereas 250-500 i.u. of vitamin D daily markedly elevated the low 25-hydroxyvitamin D concentrations.

Plasma catecholamines: laboratory aspects.

In this review the methods used for analysis of plasma catecholamines in clinical chemical laboratories are discussed. The physiology of catecholamines as well as their measuring indications are discussed, together with concise evaluation of the methods most commonly used, namely indirect radioenzymatic assays or direct determinations by high-performance liquid chromatography combined with either electrochemical or fluorometric detection. The main advantage of radioenzymatic assay is its sensitivity and thus the need for only a small sample. Liquid chromatographic methods in general are less tedious, relatively rapid, and cheap, and omit the use of radionuclides. Both of these methods, however, are subject to a number of analytical errors, which can only be avoided by proper development of methods and skilled use of these methods. Little routine work is done using either radioimmunoassay or gas-chromatography.

Determination of retinyl palmitate and total vitamin A content in liver and liver-based ready-to-eat foods.

The determination of retinyl palmitate and total vitamin A in liver and liver-based ready-to-eat foods is described. The method is very simple as sample preparation is minimal, and the isocratic elution of the C18 column with pure methanol does not necessitate a sophisticated instrumental set-up. The method is accurate with high recoveries (100.6 +/- 9.3%, mean +/- S.D., n = 23), and precise with within-day and between-day coefficients of variation of less than 5.5% (n = 13) and less than 16% (n = 6), respectively.

Immunonephelometric determination of retinol-binding protein in serum and urine.

An immunonephelometric method developed for measurement of retinol-binding protein (RBP) in serum and urine can detect it in concentrations of about 30 micrograms/L, which is in the lower limit of its normal concentration in urine (range 0-0.56 mg/L; mean +/- SD 0.19 +/- 0.15; n = 44). Urinary RBP was increased (range 0.93-29.5 mg/L) in all of 25 urine specimens from 13 subjects being treated with aminoglycoside (tobramycin). Urinary excretion of RBP was correlated (r = 0.83) with the excretion of beta 2-microglobulin. The within-assay and day-to-day precision (CV) was determined over the detection range of 0.03-8 mg/L. Within these limits the corresponding CVs varied from 4 to 27% and from 8 to 30%, respectively. The method had fairly good precision within the optimal measuring range of approximately 0.4 to 4.5 mg/L for both urine and 20-fold diluted serum samples. For various RBP concentrations our analytical recovery was 89-114% of added RBP. Results by this method correlated well (r = 0.96, n = 24) with those by a radial immunodiffusion method.

Serum albumin assay with bromcresol purple dye.

Serum albumin was assayed with bromcresol purple using routine clinical chemical photometric analysers. Bromcresol purple and bromcresol green dyes were compared, and albumin assays using these dyes were compared to those made by radial immunodiffusion and rocket immunoelectrophoresis. Bromcresol purple seems to be insensitive to all serum proteins except albumin, and this dye is correlated linearly with radial immunodiffusion and with rocket electrophoresis.

Immunonephelometric quantification of serum vitamin D-binding globulin.

Rapid quantification of serum vitamin D-binding globulin (VDBG) is described. An immunonephelometric method was developed and compared with the conventional immunodiffusion method for VDBG. A correlation coefficient of 0.896 (P less than 0.001) was found with a regression line y = 0.965x + 0.011 (n = 23). The within-assay and day-to-day coefficient of variation of the immunonephelometric method was 6 and 11% at 0.150 g/l (low VDBG concentration), 5 and 7% at 0.230 g/l (normal), and 4 and 8% at 0.570 g/l (high). Levels of VDBG and total 25-hydroxyvitamin D (25(OH)D, 25(OH)D2 + 25(OH)D3) were analysed from 33 serum samples obtained in April-May 1980 from 14-month-old children undergoing vitamin D substitution. 25(OH)D was extracted from 0.1-0.5 ml serum followed by semipreparative silica column chromatography and final quantification by a competitive protein-binding method. The VDBG mean value was 0.308 +/- 0.068 g/l (SD) and 25(OH)D had a mean value of 121 +/- 70 nmol/l (SD). There was no significant correlation between the levels of VDBG and 25(OH)D. The results of this study in general agreed well with other findings.

Determination of protein content in aqueous humour by high-performance gel filtration chromatography.

High-performance liquid chromatography (HPLC) was used to study the protein content of aqueous humour in 17 cataract patients. High-performance gel filtration chromatograms (HPGFC) of the aqueous humour showed 7-12 peaks with low concentrations (mean 0.085 micrograms/microliter) of high molecular weight proteins (peak 1, MW 250 000 or more) and low levels (0.01 micrograms/microliter or less) of IgA (peak 2) and of IgG (peak 3). The fourth peak (MW about 65 000) containing albumin and obviously also trasferrin was the major peak (mean 0.596 micrograms/microliter) at HPGFC of a normal aqueous humour. Lysozyme (peak 6, MW 35 000) occurred in small amounts in the aqueous humour (mean 0.064 micrograms/microliter). The last 6 peaks matched for peptides and amino acids (MW 10 000 or less). It is concluded that HPLC may be a useful preparative method in characterization, separation, and partial purification of immunoglobulins, immune complexes, and other proteins of aqueous humour.

Plasma haemoglobin determination using chlorpromazine as a non-carcinogenic reagent.

Plasma haemoglobin was assayed with the non-carcinogenic reagent phenothiazine. This method is sensitive and allows the measurement of plasma haemoglobin concentrations in the range 4-500 mg/l with a within-run CV of 2.1%, and a between-run CV of 4.3%. A spectrophotometric scanning method (x) based on the determination of haemoglobin as haemiglobin cyanide using the Soret band at 419 nm correlated well with the phenothiazine method (y): y = 1.07x + 15.8, r = 0.995, n = 31. It was found that the absorbances in the phenothiazine method were markedly dependent on the concentration of phosphoric acid.