Circulating levels of cytochrome C, gamma-glutamyl transferase, triglycerides and unconjugated bilirubin in overweight/obese patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease, characterized by hepatocyte apoptosis, is distinct in fatty liver and non-alcoholic steatohepatitis, the more severe form. Apoptotic cell death is caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release. Adhering to the hypothesis that the exposure of hepatocytes to free fatty acids, resulting in increased ROS production and mitochondrial damage, is balanced by the presence of antioxidant substances, circulating levels of gamma-glutamyl transferase, cytochrome c, triglycerides and unconjugated bilirubin were explored in patients suffering from non-alcoholic fatty liver disease with different severity. One hundred and eighty-six consecutive patients who presented recent ultrasound feature of bright liver without any liver disease of known origin were enrolled, eighty-nine of whom underwent liver biopsy. Forty-five subjects were allocated on the basis of histology in fatty liver group while 44 patients formed the group with non-alcoholic steatohepatitis. A cohort of 27 young, lean, apparently healthy individuals was selected as control group. The levels of gamma-glutamyl transferase were normal or slightly increased, while unconjugated bilirubin concentrations were elevated in all the spectra of non-alcoholic fatty liver disease. Comparing the present results with relevant findings from other studies dealing with diseases characterized by apoptosis, we did not find high circulating levels of cytochrome c in non-alcoholic fatty liver disease. What is more, our patients, categorized as suffering from simple fatty liver or from the more severe non-alcoholic steatohepatitis, had similar levels of cytochrome c and gamma-glutamyl transferase, p=0.19 and 0.11. Serum triglycerides were higher in patients with non-alcoholic fatty liver disease than in the healthy group, p=0.001. These findings likely reflect a balance between oxidative stress and anti-oxidant response rather than a lack of reliability of cytochrome c as a reliable biomarker of mitochondrial damage.

Does spleen volume play a role in patients with HCV-related chronic hepatitis?

As the lymphotropism of hepatitis C virus (HCV) has already been ascertained, and in the light of the fact that the immune defense system is an organized network composed of functionally interrelated tissues, this study was carried out to verify the possible involvement of spleen in HCV-related chronic hepatitis. In this cross-sectional study we measured spleen longitudinal diameter by ultrasound, beta2-microglobulin serum levels and splenic artery resistivity index (SARI) by Doppler in 51 patients treated with standard combined (Peg-Interferon plus Ribavirin) antiviral therapy. Thirty-three patients (17 females) completed the regimen and were compared to 31 controls (16 females). The mean basal values of spleen longitudinal diameter were higher in patients with chronic hepatitis than in controls, i.e., 116 mm (9.4) versus 102.7 mm (9.3), P = 0.0001. In the same patients a significant trend towards increased spleen longitudinal diameter was found after antiviral therapy, independently of the stage of HCV-related chronic hepatitis. The median values of the beta2-microglobulin concentrations were not significantly higher in the patients with HCV-related chronic hepatitis compared to controls, i.e., 1.3 (0.5-2.6) versus 1 (0.6-1.4), P = 0.16, although during the course of therapy they were significantly increased. SARI values of HCV-related chronic hepatitis patients were different from those of controls, but were unvaried compared to values at the end of treatment. Neither spleen measurements nor serum beta2-microglobulin levels were able to predict therapeutic response to antiviral therapy. A stimulation/expansion of lymphoid tissue was found in patients with HCV-related chronic hepatitis. Such evidence raises the question whether physicians should continue to prescribe antiviral therapy in non-responders and supports the use of a new scheme (SLD plus beta2-MG) to diagnose this ongoing, apparently reversible, but nevertheless dangerous immunologic process.

Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

Cardiac performance during exercise in patients with Fabry's disease.

BACKGROUND: Fatigability and dyspnoea on effort are present in many patients with Fabry's disease. We assessed the determinants of cardiac performance during exercise in patients with Fabry's disease and preserved left ventricular ejection fraction at rest. MATERIALS AND METHODS: Sixteen patients with Fabry's disease and 16 control subjects underwent radionuclide angiography at rest and during exercise, tissue Doppler echocardiography and magnetic resonance imaging at rest. RESULTS: The exercise-induced change in stroke volume was +25 +/- 14% in controls and +5.8 +/- 19% in patients with Fabry's disease (P < 0.001). In 10 patients (group 1), the stroke volume increased (+19 +/- 10%), and in 6 patients (group 2) it decreased (-16 +/- 9%) with exercise. Patients of group 2 were older, had worse renal function, higher left ventricular mass and impaired diastolic function compared to group 1. The abnormal stroke volume response to exercise in group 2 was associated with a decrease in end-diastolic volume (P < 0.001) and a lack of reduction of end-systolic volume (P < 0.01) compared with both controls and group 1. The ratio of peak early-diastolic velocity from mitral filling to peak early-diastolic mitral annulus velocity was the only independent predictor of exercise-induced change in stroke volume (B -0.44; SE 0.119; beta-0.70; P < 0.005). CONCLUSIONS: The majority of patients with Fabry's disease were able to augment stroke volume during exercise by increasing end-diastolic volume, whereas patients with more advanced cardiac involvement may experience the inability to increase cardiac output by the Frank Starling mechanism.

Relationship between calcium absorption and plasma dehydroepiandrosterone sulphate (DHEAS) in healthy males.

CONTEXT: Impaired gut sensitivity to 1,25-dihydroxyvitamin D (1,25(OH)(2)D), leading to reduced intestinal calcium absorption, has been reported in older men and women. While this phenomenon in postmenopausal women has been attributed to oestrogen deficiency, it is unclear whether the same observation in older men correlates with the age-related decline in androgen concentrations. OBJECTIVE: To examine the relationship between androgens and intestinal calcium absorption in older men. DESIGN: Cross-sectional study on 55 healthy male volunteers, divided into younger (n = 27) and older (n = 28) groups separated according to the median age of 59 years. MAIN OUTCOME MEASURES: Calcium absorption, total and free (calculated) testosterone, dehydroepiandrosterone sulphate (DHEAS), SHBG, and 1,25(OH)(2)D, among others, were measured. RESULTS: Calcium absorption, free testosterone and DHEAS, but not 1,25(OH)(2)D, declined significantly with age. After adjusting for age and body mass index, stepwise regression showed that 1,25(OH)(2)D and serum albumin were the only significant determinants of calcium absorption in younger men, while the sole determinant in older men was DHEAS, not testosterone. Residual deviations from the regression of calcium absorption on 1,25(OH)(2)D, reflecting the efficiency of 1,25(OH)(2)D-induced calcium absorption, was positively correlated with DHEAS (r = 0.27, P = 0.027). CONCLUSIONS: DHEAS is an independent determinant of calcium absorption in older men, although its manner of influence is, as yet, undefined. The age-related decline of DHEAS may, partly, account for the observed 'intestinal resistance to 1,25(OH)(2)D' in older men.

Evaluation of the monocyte counting by two automated haematology analysers compared with flow cytometry.

The aim is to determine the monocyte count performance of the Bayer Diagnostics ADVIA120 and Coulter LH 750 automated haematology analysers and the results obtained by these two instruments were compared with those provided by Becton Dickinson FACScan flow cytometer using the combination of CD45/CD14 MoAb. Linearity and imprecision were also evaluated. The linearity of both instruments was good. Coulter LH 750 showed better precision (4.3%) than ADVIA 120 (9.0%) both within and between batch. A significant correlation (r = 0.973) was found between the LH 750 and the flow cytometry method, while a modest one was observed between the latter and the ADVIA 120 (r = 0.880). When comparing the percentage of monocytes by means of one-way anova and Tukey test, it was found that the LH 750 provided the closest results in comparison with flow cytometry, with no statistical difference between the means (mean difference MO% = 0.6); however the difference was statistically different between the ADVIA 120 and flow cytometry (mean difference MO% = -4.06). These data were confirmed by Altman-Bland and Deming regression analyses.

[The fluid balance in the critically ill patient]. / Il bilancio idrico nel malato di terapia intensiva.

AIM: In the critically ill patient the hemodynamic management is essential. Usually the hemodynamic status is estimated by the measurement of the intravascular pressure or volume and by computing the fluid balance, while the change in body weight estimates the total body water. The fluid balance is computed by subtracting the fluid output to the input. Although the input (fluids, drugs, infusion.) are well known, the output are difficult to correctly compute, because they depend on several factors such as the body-room temperature, amount of humidity and may change with time. Aim of this study was to prospectively evaluate the accuracy of the fluid balance compared to the body weight measured by a dedicated mattress (Hill-Rom). METHODS: Fifteen critically ill patients were enrolled (mean age of 63.2+/-19.7 years, body mass index of 24.9+/-3.5 kg/m2) and the measurements were computed every 8 hours. The data were analyzed by the Bland-Altman test. RESULTS: Four-hundred and seventy-two measurements for a mean intensive care stay of 12.3.+/-7.3 days were computed. The Bland-Altamn showed a great inaccuracy, because the agreement (the difference between the 2 methods) was poor with a standard deviation of 1 840 L. CONCLUSION: Although the fluid balance was not accurate, the only body weight without any intravascular measurement is not helpful for a correct clinical hemodynamic management of the patient.

Relation between calcium absorption and serum calcitriol in normal men: evidence for age-related intestinal resistance to calcitriol.

OBJECTIVE: To obtain information on the causes of age-related bone loss in men and the concomitant decline in calcium absorption. DESIGN: Cross-sectional study. SETTING: Adelaide, South Australia, Australia. SUBJECTS: A total of 95 healthy, Caucasian men (age range 27-87 y). RESULTS: Calcium absorption declined with age (r=-0.46, P<0.0001), as did 24-h urine calcium, phosphate and creatinine (r>-0.21, P<0.05 for all); serum calcitriol and 25 hydroxyvitamin D did not change with age. Calcium absorption was related to serum calcitriol (r=0.20, P=0.05). An inverse relation between the residual deviations in calcium absorption, after allowing for its dependence on calcitriol, and age (F=5.4, P<0.005) was observed. The 24-h urinary calcium, phosphate and creatinine were all related to calcium absorption (r>0.41, P<0.0001). Forearm bone density fell with age (r=-0.45, P<0.0001) but was not related to calcium absorption, or markers of bone turnover. CONCLUSIONS: In healthy Caucasian males (i) calcium absorption falls, but serum calcitriol does not change with age, (ii) the relation between calcium absorption and serum calcitriol changes with age, indicative of an intestinal resistance to calcitriol and (iii) calcium absorption is a significant determinant of 24-h urinary calcium excretion.

Association between vitamin D receptor gene polymorphisms and tubular citrate handling in calcium nephrolithiasis.

OBJECTIVES: Hypocitraturia is a risk factor for calcium nephrolithiasis. 1,25(OH)2D3 influences renal citrate handling and enhances citraturia. The aim of this study was to evaluate the relationship between vitamin D receptor (VDR) allelic variant and urinary citrate excretion in recurrent stone formers (SF) patients. DESIGN: Case-control study. SUBJECTS: A total of 220 recurrent calcium oxalate SF patients and 114 healthy control (C) subjects were enrolled for this study. Subjects with urinary tract infections, hyperparathyroidism, cystinuria >70 micromol/24 h, gouty diathesis, renal tubular acidosis, renal failure, chronic diarrhoeal states, intake of thiazide diuretics, angiotensin-converting enzyme (ACE)-inhibitors, glucocorticoids or oestrogens were excluded. A standard constant diet was given for 7 days. The 24-h urinary citrate excretion and the active tubular reabsorption of filtered citrate (Rcit) were evaluated. Hypocitraturia was defined as a urinary citrate excretion lower than 1.7 mmol day-1. Stone formers patients and C were genotyped for BsmI and TaqI VDR alleles. Contingency table chi-square tests were used to compare genotype frequencies in hypocitraturic SF patients, normocitraturic SF and C. RESULTS: The prevalence of hypocitraturia in SF patients was 32.7% (72 of 200). Hypocitraturia in these patients resulted from excessive Rcit of a normal load of citrate. We found a different distribution (P < 0.05) of BsmI and TaqI VDR genotypes in hypocitraturic SF patients compared with normocitraturic SF and C. In particular, the prevalence of bb and TT VDR genotypes in hypocitraturic SF was significantly higher than in normocitraturic SF and C. CONCLUSIONS: These results point to a genetic association between BsmI and TaqI VDR polymorphisms and idiopathic hypocitraturia in calcium-oxalate recurrent SF patients.

Bone density and bone-related biochemical variables in normal men: a longitudinal study.

BACKGROUND: The objective of this study was to determine the pattern of forearm bone loss and its relationship to markers of bone turnover and sex steroids in normal men. This was a longitudinal study over a median interval of 41 months. The study was conducted in Adelaide, Australia. Study participants were 123 healthy male subjects, between the ages of 20 and 83 years. METHODS: Fat-corrected forearm bone mineral content (fcBMC), markers of bone formation (alkaline phosphatase, osteocalcin, procollagen type 1 C-terminal extension peptide) and bone resorption (collagen type I cross-linked telopeptide, hydroxyproline/creatinine, pyridinoline/creatinine, and deoxypyridinoline/creatinine), calculated serum bioavailable testosterone, and serum estradiol were measured. RESULTS: The mean time-weighted rate of change in forearm fcBMC was -0.33% +/- 0.72 (SD) per year. Bone loss commenced after 30 years of age and increased with age (p <.001), particularly after age 70 years. There was no relationship between the rate of change in fcBMC and either markers of bone turnover or serum sex steroids. CONCLUSIONS: In normal men, bone loss increases with age; there does not appear to be any relationship between this loss and either markers of bone turnover or levels of free androgen or estrogen.

Exercise increases soluble adhesion molecules ICAM-1 and VCAM-1 in patients with intermittent claudication.

Adhesion molecules play a relevant role in the pathogenesis of vascular diseases. In 21 patients with intermittent claudication and 18 sex- and age-matched control subjects, we measured plasma levels of the circulating form of the adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) alongside von Willebrand factor (vWF), at rest, at maximally tolerated exercise and 5, 15 and 30 min after exercise. In controls, plasma sICAM-1 levels did not change with exercise, while in claudicants they increased from 285+/-15 to 317+/-16 ng/ml (p<0.01). Also for sVCAM-1 exercise did not modify plasma levels of sVCAM-1 in controls but increased it in claudicants from 671+/-45 to 751+/-47 ng/ml (p<0.05). Similarly, vWF did not change with exercise in controls, but increased in claudicants from 100+/-9% to 111+/-8% of value for pooled normal plasma (p<0.05). Exercise-induced changes in sICAM-1 negatively correlated with the maximal tolerated walking time, which is an index of disease severity. These findings indicate that, in claudicants, exercise is associated with increase in plasma levels of sICAM-1 and sVCAM-1.

High levels of adhesion molecules are associated with impaired endothelium-dependent vasodilation in patients with peripheral arterial disease.

Soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cellular adhesion molecule-1 (sVCAM-1) were measured alongside flow-mediated vasodilation (FMD) in 34 patients with intermittent claudication and 14 control subjects. Patients with plasma sICAM-1 >253 ng/mL (median value) showed lower FMD than those with sICAM-1 < 253 ng/mL (5.6 +/- 1.8% vs 9.6 +/- 4.2%, p < 0.01). Similarly, in the 17 patients with plasma sVCAM-1 > 414 ng/mL, FMD was lower than in the remaining 17 patients (6.1 +/- 1.9% vs 9.2 +/- 4.5%, p < 0.05). Additionally, when endothelial dysfunction was defined as FMD < or = 5.5%, patients with FMD below this value had higher plasma concentrations of sICAM-1 and sVCAM-1 than those with FMD > 5.5%. Therefore, our findings indicate a close association between elevated plasma levels of adhesion molecules and endothelial dysfunction. As impaired endothelial function is one of the first steps in atherogenesis, our findings have clinical relevance since they serve as the basis for further evaluation of sICAM-1 and sVCAM-1 as potential plasma markers for progression of atherosclerosis in a population at high risk.

Circulating levels of cytokines and their site of production in patients with mild to severe chronic heart failure.

BACKGROUND: Patients with chronic heart failure have elevated levels of proinflammatory cytokines; however, the mechanism for their increased expression and the site of their production are unknown. METHODS: Twenty-two patients with heart failure, New York Heart Association functional class II to IV, underwent hemodynamic evaluation and echocardiographic study. Blood samples for cytokine evaluation were performed in the ascending aorta, coronary sinus, inferior vena cava, and hepatic vein. Levels of tumor necrosis factor-alpha (TNF-alpha), its soluble receptors sTNF-RI and sTNF-RII, interleukin-6 (IL-6), IL-6 soluble receptor, soluble gp130, interleukin-2 soluble receptor, and soluble Fas were measured with enzyme-linked immunosorbent assay kits. RESULTS: IL-6 concentrations were higher in class IV patients than in class III patients, which in turn were higher than those in class II. TNF-alpha, sTNF-RI, and sTNF-RII were higher in class IV patients than in class III and II patients. Significant correlations were found between IL-6 concentrations and left ventricular end-systolic volume (r = 0.64; P <.001), pulmonary wedge pressure (r = 0.56; P <.01), and left ventricular ejection fraction (r = -0.56; P <.01). No correlation was found between TNF-alpha and its soluble receptors and left ventricular volumes or hemodynamic measures. Finally, no difference in cytokine concentrations was found among the different sample sites. CONCLUSIONS: Among inflammatory cytokines, IL-6 concentrations better reflect the hemodynamic derangement in patients with heart failure. No cardiac or gut production of cytokines occurs in patients with mild to severe heart failure.

Measurement of soluble adhesion molecules in primary Raynaud's phenomenon and in Raynaud's phenomenon secondary to connective tissue diseases.

Adhesion molecules play a role in the inflammation and pathogenesis of vascular diseases. In 13 patients with primary Raynaud's phenomenon, 19 with Raynaud's phenomenon associated with connective tissue disease, and 16 control subjects, we measured plasma levels of soluble forms of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and von Willebrand factor. Patients with secondary Raynaud's phenomenon had plasma levels of soluble forms of intercellular adhesion molecule- 1, vascular cell adhesion molecule-1, E-selectin, and von Willebrand factor which were significantly higher than in those with primary Raynaud's phenomenon and controls, while no difference was observed between patients with primary Raynaud's phenomenon and controls. Within the group with secondary Raynaud's phenomenon, the strongest correlations were between soluble forms of intercellular adhesion molecule-1 and both E-selectin, (r=0.67, P<0.001) and von Willebrand factor (r=0.58, P<0.01). In none of the three groups were the levels of soluble adhesion molecules and von Willebrand factor changed by exposure of hands to cold, although all patients had a definite vasospasm. In conclusion, this study indicates that primary Raynaud's phenomenon is not associated with elevation of soluble adhesion molecules and von Willebrand factor. Prospective studies are now required to investigate the role of these molecules as predictors of secondary diseases.

The relation between bone density, free androgen index, and estradiol in men 60 to 70 years old.

The cause of age-related bone loss in men is poorly understood. Previous studies of the relationship between bone density and serum androgens have yielded inconsistent results, perhaps partly because age is a determinant of both. Recent studies suggest that serum estrogen levels influence bone density in adult men. In order to determine whether bone mineral density (BMD) and bone turnover are associated with serum sex steroids, we investigated 37 normal men within a narrow age range (60-70 years). Bone mineral density at the forearm, hip, and spine, testosterone, sex hormone binding globulin (SHBG), free androgen index (FAI:T/SHBG), estradiol (E), free estradiol index (FEI:E/SHBG), and markers of bone formation (alkaline phosphatase, osteocalcin, procollagen type I C-terminal extension peptide) and bone resorption (hydroxyproline/creatinine [OHPr/Cr], deoxypyridinoline/creatinine [Dpd/Cr], pyridinoline/creatinine, collagen type I cross-linked telopeptide) were measured. Bone mineral density was positively related (r > 0.35, p < 0.05 at all sites) to log FAI, whereas there was no significant relationship between BMD and either serum total testosterone, serum E, or FEI. Bone density at the spine and hip were inversely related to both OHPr/Cr (r > -0.41, p < 0.05 for all sites) and Dpd/Cr (r > -0.36, p < 0.05 for all sites). OHPr/Cr (r = -0.41, p < 0.05) and Dpd/Cr (r = -0.41, p < 0.05) were both inversely related to log FAI. We conclude that BMD and bone turnover in adult men are related to plasma free androgens.

Blood pressure and metabolic changes during dietary L-arginine supplementation in humans.

Dietary L-arginine supplementation has been proposed to reverse endothelial dysfunction in such diverse pathophysiologic conditions as hypercholesterolemia, coronary heart disease, and some forms of animal hypertension. In particular, chronic oral administration of L-arginine prevented the blood pressure rise induced by sodium chloride loading in salt-sensitive rats. To investigate the effects of L-arginine-rich diets on blood pressure and metabolic and coagulation parameters we performed a single-blind, controlled, crossover dietary intervention in six healthy volunteers. The subjects (aged 39+/-4 years, body mass index [BMI] 26+/-1 kg/m2, mean +/- SEM) received, in random sequence, three different isocaloric diets, each for a period of 1 week (Diet 1: control; Diet 2: L-arginine enriched by natural foods; Diet 3: identical to Diet 1 plus oral L-arginine supplement). Sodium intake was set at a constant level (about 180 mmol/day) throughout the three study periods. A blood pressure decrease was observed with both L-arginine-rich diets (Diet 2 v 1, SBP: -6.2 mm Hg [95% CI: -0.5 to -11.8], DBP: -5.0 mm Hg [-2.8 to -7.2]; Diet 3 v 1, SBP: -6.2 mm Hg [-1.8 to -10.5], DBP: -6.8 mm Hg [-3.0 to -10.6]). A slight increase in creatinine clearance (P = .07) and a fall in fasting blood glucose (P = .008) occurred after Diet 3 and, to a lesser extent, after Diet 2. Serum total cholesterol (P = .06) and triglyceride (P = .009) decreased and HDL cholesterol increased (P = .04) after Diet 2, but not after Diet 3. These results indicate that a moderate increase in L-arginine significantly lowered blood pressure and affected renal function and carbohydrate metabolism in healthy volunteers.

Evaluation of the Abbott CELL-DYN 4000 hematology analyzer.

A new generation hematology analyzer, Abbott CELL-DYN 4000 (CD 4000), capable of providing 26 parameters, including fully automated reticulocyte, nucleated RBC, blast, band, and immature granulocyte, and variant lymphocyte counts, was evaluated by using the National Committee for Clinical Laboratory Standards H20-A protocol and compared with the Bayer-Technicon H-2 analyzer, which is used routinely in our laboratory. A lipid interference experiment and a sample aging study also were performed. Linearity, carryover, and precision were within the limits established by the manufacturer, and satisfactory agreement was found with the H-2 analyzer. The evaluation of leukocyte differential counts indicated an excellent correlation with the manual reference method for neutrophils and lymphocytes, a good correlation for monocytes and eosinophils, and a poor correlation for basophils in samples with low counts; for basophil counts of 2% or higher, we found an improvement of the correlation coefficient. In the lipid interference experiment, only hemoglobin determination was influenced significantly on the CD 4000, but by using a new Abbott hemoglobin reagent, the interference was eliminated. The CBC and differential counts were stable and reportable up to at least 24 hours. Intrasample viability information on leukocytes provided a quality check on each individual specimen.

Inhibition of bone resorption by divided-dose calcium supplementation in early postmenopausal women.

We have previously shown that a calcium (Ca) supplement of 1000 mg given in the evening reduces the overnight and early morning, but not the daytime, excretion of bone resorption markers in postmenopausal women within five years of the menopause. In the present study, we have looked at the effect of splitting the Ca into two doses of 500 mg each given in the morning and evening. We studied 19 healthy women (median age 53 years) who were all within 5 years of the menopause. On the 2 study days, urine was collected from 9 a.m. to 9 p.m. (day collection), and from 9 p.m. to 9 a.m. (night collection); a further fasting (spot) urine sample was obtained at 9 a.m. at the end of the night collection. The first day was a control day; on the second day the subjects ingested 500 mg Ca as the carbonate at 9 a.m. and 9 p.m. We measured pyridinoline cross-links excretion in all the samples, as well as hydroxyproline in the fasting urine. The Ca supplements lowered urinary excretion of the markers during the day (P < 0.01), had only a marginal effect during the night, but reduced excretion significantly in the fasting urine (P < 0.001). In the whole 24-hour period, the falls in resorption markers were small but comparable to those seen after the ingestion of 1 g of Ca in the evening. We conclude that the acute administration of 0.5 g Ca in the morning and evening reduced the markers of bone resorption in early postmenopausal women during the day but not during the following night, whereas the single 1 g supplement had the reverse effect. Over the 24-hour period, there was nothing to choose between the two regimes. Women at this stage in their life cycle probably require a larger Ca supplement if they are not taking estrogen.