Noninvasive markers of oxidative DNA stress, RNA degradation and protein degradation are differentially correlated with resting metabolic rate and energy intake in children and adolescents.

Urinary excreted RNA and DNA catabolites are used as noninvasive markers for metabolic processes: 8-oxo-2'-deoxyguanosine (8-oxodG) potentially represents oxidative stress to DNA/deoxyribonucleotidetriphosphate pool, modified ribonucleoside pseudouridine (psi) originating mainly from degraded rRNA and tRNA reflects RNA turnover. Modified amino acid gamma-carboxyglutamic acid (Gla) stems from degraded proteins reflecting turnover of proteins. Aim of the present study was to investigate (44 healthy males, 3-18 y) how excretion rates of 8-oxodG, psi, and Gla are related to resting metabolic rate and energy intake. Excretion rates of 8-oxodG (pmol/kg/d), psi (micromol/kg/d), and Gla (micromol/kg/d) were significantly correlated with resting metabolic rate (kJ/kg/d): r = 0.108 (p = 0.029), 0.691 and 0.552 (p < 0.0001), respectively. Excretion rates of 8-oxodG, psi, and Gla were also significantly correlated with energy intake (kJ/kg/d): r = 0.108 (p = 0.036), 0.602 and 0.462 (p < 0.0001). 8-oxodG and Gla excretion was significantly correlated with psi excretion: r = 0.174 (p = 0.005) and 0.709 (p < 0.0001). These results indicate close relationships between whole-body RNA and protein degradation and metabolic rate. The relationship between 8-oxodG excretion and metabolic rate, however, is less strong suggesting that factors other than metabolic rate considerably affect the oxidative stress to DNA.

Effect of phylloquinone supplementation on biochemical markers of vitamin K status and bone turnover in postmenopausal women.

While current intakes of phylloquinone (vitamin K1) in many populations are believed to be sufficient to maintain normal blood coagulation, these may be insufficient to cover the requirements for optimal bone metabolism. Therefore, the objective of the present study was to investigate the effect of increasing phylloquinone intakes above the usual dietary intake for 6 weeks on biochemical markers of vitamin K status and bone turnover in postmenopausal women. Thirty-one postmenopausal women completed this 3 x 6-week randomised cross-over study, in which volunteers were supplemented with 0 (placebo), 200, and 500 microg phylloquinone/d. In addition, the volunteers were given 10 microg vitamin D3/d throughout the study period. With increasing phylloquinone intake, the concentration of serum gamma-carboxylated and under-gamma-carboxylated osteocalcin was significantly increased and decreased, respectively, in a dose-dependent manner (P < 0.001). Mean serum phylloquinone concentration was significantly (P < 0.001) higher with daily supplementation with 500 microg phylloquinone/d compared with that during either of the placebo or 200 microg phylloquinone/d supplementation periods, which did not differ (P = 0.15). Serum total osteocalcin was significantly (P < 0.001) increased in response to daily supplementation with 500 (but not 200) microg phylloquinone compared with placebo. Serum bone-specific alkaline phosphatase as well as the urinary markers of bone resorption (N-telopeptide cross-links of collagen, pyridinoline and deoxypyridinoline) and urinary gamma-carboxyglutamate were unaffected by phylloquinone supplementation. In conclusion, while daily supplementation with 200 and 500 microg phylloquinone/d for 6 weeks increased vitamin K status in postmenopausal women, it had no effect on bone turnover.

Dietary phylloquinone depletion and repletion in older women.

Biological markers indicative of poor vitamin K status have been associated with a greater risk for hip fracture in older men and women. However, the dietary phylloquinone intake required to achieve maximal carboxylation of hepatic and extrahepatic vitamin K-dependent proteins is not known. In an 84-d study in a metabolic unit, 21 older (60-80 y) women were fed a phylloquinone-restricted diet (18 micro g/d) for 28 d, followed by stepwise repletion of 86, 200 and 450 micro g/d of phylloquinone. Plasma phylloquinone, urinary gamma-carboxyglutamic acid excretion and gamma-carboxylation of hepatic (prothrombin) and extrahepatic proteins (osteocalcin) decreased in response to phylloquinone restriction (P < 0.001), demonstrating the production of subclinical vitamin K deficiency. The gamma-carboxylation of prothrombin was restored to normal levels in response to phylloquinone supplementation at 200 micro g/d. In contrast, all other biochemical markers of vitamin K status remained below normal levels after short-term supplementation of up to 450 micro g/d of phylloquinone. These data support previous observations in rats that hepatic vitamin K-dependent proteins have preferential utilization of phylloquinone in response to phylloquinone dietary restriction. Moreover, our findings suggest that the current recommended Adequate Intake levels of vitamin K (90 micro g/d) in women do not support maximal osteocalcin gamma-carboxylation in older women.

Effects of skeletal loading on bone mass and compensation mechanism in bone: a new insight into the "mechanostat" theory.

We have suggested that: (i) osteocalcin carboxylation is related to bone material properties (bone quality), and (ii) impairment of bone material properties could be compensated by bone mass increase. The aim of the present prospective study was to investigate whether the effects of skeletal loading on bone mass were associated with the compensation mechanism between bone mass and bone material properties. The subjects were 56 healthy female volunteers aged around 50 years. They were classified into pre- and postmenopausal groups, and each group was then subdivided into a no-exercise (control) and a vertical jumping exercise group. Bone mineral density (BMD) was measured at baseline and after the 6-month study period. Urinary gamma-carboxyglutamate (Gla), a possible parameter of osteocalcin carboxylation, was also measured at baseline. Among the premenopausal women, hip BMD in the exercise group increased significantly in comparison to the control value. Among the postmenopausal women, however, there was no significant difference in the BMD change between the control and the exercise group. In addition, the baseline urinary Gla level showed an inverse correlation with the change in whole body BMD in the premenopausal exercise group. These results suggest that: (i) estrogen plays a certain role in the high-impact exercise-induced bone gain, and (ii) the effects of skeletal loading on bone mass are involved in the compensation mechanism, i.e., bone gain due to high-impact exercise becomes greater in accordance with the degree of deterioration in bone material properties. Our concept of the compensation mechanism could provide a new insight into the understanding of the skeleton's adaptability to load-bearing.

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence.

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.

Gender differences in hepatic phylloquinone and menaquinones in the vitamin K-deficient and -supplemented rat.

Gender differences in relation to vitamin K were investigated in the rat. Hepatic phylloquinone and menaquinone (MK-1 to MK-10) concentrations, gamma-carboxyglutamic acid (Gla) excretion, plasma phylloquinone and percent prothrombin were measured in male and female rats on a chow diet (24.5 ng phylloquinone and 8.8 microgram menadione), and on phylloquinone-deficient and -supplemented purified diets (0.38 and 1400 ng phylloquinone/g, respectively). Mean hepatic phylloquinone concentrations varied with dietary intake and ranged from 6.8+/-9.0 pmol/g in the deficient male, to 171. 1+/-56.9 pmol/g in the supplemented female. Menaquinones accounted for a large proportion of total vitamin K in the liver of males and females with MK-4, MK-6, and MK-10 present in highest concentrations. On the chow and supplemented diets, females had significantly higher MK-4, MK-6, and MK-10 concentrations in their livers (P<0.05). On the phylloquinone-deficient diet (-K1), hepatic phylloquinone, MK-4, and to a lesser extent MK-6 (but not MK-10) were significantly reduced (P<0.05). In the phylloquinone-supplemented male and female groups, which did not receive menadione during the experimental period, MK-4 increased above that in the chow groups suggesting synthesis of MK-4 from phylloquinone which was statistically significant in the female (P<0.01). A significant gender difference (P<0.05) was also observed for urinary Gla excretion with less Gla excreted by the females indicating that females may require less dietary phylloquinone than males of the same body weight.

Interaction between vitamin K nutriture and bacterial overgrowth in hypochlorhydria induced by omeprazole.

Subjects taking a hydrogen pump blocking agent (omeprazole) develop bacterial overgrowth of the small intestine. We tested the hypothesis that this bacterial overgrowth produces menaquinones, which would meet the vitamin requirement in situations of vitamin K deficiency. In a crossover-type design, 13 healthy volunteers eating a phylloquinone-restricted diet for 35 d were randomly assigned to take omeprazole during the first period of study or starting on day 15 until the end of the study. Coagulation times, serum osteocalcin [total osteocalcin and undercarboxylated osteocalcin (ucOC)], plasma phylloquinone, urinary gamma-carboxyglutamic acid, and plasma undercarboxylated prothrombin (PIVKA-II) were measured. Plasma phylloquinone concentrations declined 82% with dietary phylloquinone restriction (P < 0.05) and were not significantly different in the period when the diet was combined with omeprazole treatment (P > 0.05). The mean value for PIVKA-II during the phylloquinone-restricted diet significantly increased 5.7-fold from baseline (P < 0.05); however, the combination of omeprazole treatment and the phylloquinone-restricted diet significantly reduced PIVKA-II values by 21% (P < 0.05) compared with the diet period alone. There were no alterations in total or percentage ucOC concentrations during the phylloquinone-restricted diet or during the period of diet plus omeprazole treatment. Our data support the hypothesis that bacterial overgrowth results in the synthesis and absorption of menaquinones. These menaquinones contribute to vitamin K nutriture during dietary phylloquinone restriction, but not enough to restore normal vitamin K status.

Bone markers during a 6-month space flight: effects of vitamin K supplementation.

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.

Effect of vitamin K1 supplementation on vitamin K status in cystic fibrosis patients.

BACKGROUND: Patients with cystic fibrosis are at risk for impaired vitamin K status due to fat malabsorption from pancreatic insufficiency. This study was designed to assess vitamin K status and measure the effect of vitamin K1 supplementation in cystic fibrosis patients. METHODS: Eighteen outpatients participated in a crossover study to determine the effect of vitamin K1 (phylloquinone) supplementation. After obtaining initial data, each subject was randomly assigned to either a 4-week study treatment of 5 mg oral vitamin K1 supplementation per week, or no supplementation and then crossed over to the other treatment for a second 4 week period. Plasma, serum and urine samples were collected and analyzed pre-study and at the end of each study period. RESULTS: The mean concentration of plasma vitamin K1 for the supplemented group was significantly higher than the unsupplemented group, [0.34 nmol/L and 0.21 nmol/L, respectively (p < 0.05)]. The percent of undercarboxylated osteocalcin increased on supplementation from 17% to 31%, (p < 0.005). Prothrombin induced in vitamin K absence (PIVKA-II) increased on supplementation from 5 ng/mL to 22 ng/mL, (p < 0.005). The ratio of urinary gamma-carboxyglutamic acid/creatinine was similar for both study periods. CONCLUSIONS: In contrast to other studies in cystic fibrosis, this study demonstrated a need for vitamin K1 supplementation. The carboxylation state of osteocalcin and PIVKA-II were the most sensitive indices of changes in vitamin K1 status. Although the 5 mg vitamin K1/week dose improved these vitamin K parameters, normal levels were not achieved.

Changes in serum osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid in response to altered intakes of dietary phylloquinone in human subjects.

The response of osteocalcin and other biochemical markers of vitamin K status to diets formulated to contain different amounts of phylloquinone was assessed in nine healthy subjects aged 20-33 y. Subjects resided in a metabolic ward for two 15-d cycles with a minimum of 6 wk between cycles. A mixed diet containing 100 micrograms phylloquinone/d was fed throughout both cycles; however, the phylloquinone content of one of the cycles was increased to a total of 420 micrograms/d on days 6 through 10 by fortifying corn oil in the diet with phylloquinone (supplemented diet). Total serum osteocalcin concentrations were not affected by either of the dietary treatments. The percentage of undercarboxylated osteocalcin increased an average of 28% over the 15-d cycle with the mixed diet (P < 0.05) and declined significantly an average of 41% with 5 d of the supplemented diet (day 6: 21.9 +/- 1.3%, day 11: 12.8 +/- 1.1%; P = 0.0001) with a rise after the return to the mixed diet (16.7 +/- 1.3%, P < 0.001). Plasma phylloquinone concentrations increased significantly with supplementation (day 6: 0.95 +/- 0.16 nmol/L, day 11: 1.40 +/- 0.29 nmol/L; P < 0.001) and then rapidly returned to presupplementation concentrations on returning to the mixed diet. Twenty-four-hour ratios of urinary gamma-carboxyglutamic acid to creatinine were unchanged with the supplemented diet; however, excretion declined to 91 +/- 2% of baseline after 10 d on the mixed diet (P = 0.01). These results show that undercarboxylated osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid excretion appear to be sensitive measures of vitamin K nutritional status because all of these variables were responsive to changes in dietary intake.

Nutritional vitamin K-intake and urinary gamma-carboxyglutamate excretion in the rat.

Using the rat as an experimental animal model we have found that prothrombin synthesis reaches its maximal level at a relatively low dietary vitamin K intake. At still higher vitamin K intakes, however, the urinary Gla-excretion was substantially increased, showing a different vitamin K requirement for liver and extrahepatic tissues. The increased urinary Gla-excretion was found for both phylloquinone and menaquinone-4, but not for menaquinone-8, which questions the bioavailability of higher menaquinones for extrahepatic tissues. A discrepancy was found between effects of nutritional vitamin K-deficiency and treatment with a vitamin K-antagonist (brodifacoum). With both regimens plasma prothrombin rapidly decreased to well below 10% of the starting values, but in case of K-deficiency urinary Gla had hardly decreased in 7 days, whereas after 3 days of brodifacoum treatment Gla-excretion had decreased to 17% of the starting values. An explanation for this observation is that prothrombin procoagulant activity does not decrease proportional to the prothrombin Gla-content, but that a wide range of undercarboxylated prothrombins have lost nearly all activity. During vitamin K-deficiency the remaining low levels of vitamin K would mainly give rise to undercarboxylated prothrombin, whereas during brodifacoum treatment only non-carboxylated prothrombin is formed. It seems plausible that in the latter case the urinary Gla originates from proteins with long half-life times, such as the bone Gla-proteins.

Assessment of vitamin K status in human subjects administered "minidose" warfarin.

Vitamin K is required to convert specific glutamyl residues in a limited number of proteins to gamma-carboxyglutamyl residues. The response of various measures of vitamin K insufficiency to the administration of 1 mg/d of the vitamin K antagonist warfarin was studied in two groups of nine older (55-75 y) or younger (20-28 y) subjects. The most consistent and extensive alteration was an increase in the concentration of serum under-gamma-carboxylated osteocalcin followed by increased immunochemical detection of plasma under-gamma-carboxylated prothrombin (PIVKA-II), and by a decreased urinary excretion of gamma-carboxyglutamic acid. Plasma concentrations of prothrombin were altered by this treatment but prothrombin times, factor VII activity, prothrombin F-1 x 2 concentrations, and a less sensitive assay for under-gamma-carboxylated prothrombine were not. The concentration of serum under-gamma-carboxylated osteocalcin was lower when subjects consumed 1 mg vitamin K/d than when they consumed their normal diet.

Comparison of biochemical indexes for assessing vitamin K nutritional status in a healthy adult population.

Biochemical indexes for assessing vitamin K nutritional status were evaluated in 263 healthy subjects (127 males, 136 females) aged 18-85 y. The influences of aging (stratified by decade), menopause, and sex were examined. Total, carboxylated, and undercarboxylated osteocalcin concentrations were affected by sex and aging with increases in the sixth decade in women attributed to menopause. Aging effects in the women and sex differences were eliminated when undercarboxylated osteocalcin was expressed as a percentage of the total. Plasma phylloquinone and undercarboxylated prothrombin (PIVKA-II) concentrations varied little with aging with the exception of lower concentrations of phylloquinone in women in their third decade compared with other ages and higher concentrations of PIVKA-II in younger males compared with younger females. Urinary gamma-carboxyglutamic acid (Gla)-creatinine excretion ratios increased significantly with age in both males (r = 0.68, P < 0.001) and females (r = 0.63, P < 0.001) with values 20% higher in the females on average over the entire age span. The undercarboxylated osteocalcin concentration, shown previously to be responsive to depletion and repletion of phylloquinone, was compared with the other indexes to determine its reliability as an indicator of vitamin K nutritional status. This measure appears promising because it correlated with plasma PIVKA-II concentrations (r = 0.27, P < 0.001) and with plasma phylloquinone concentrations (r = -0.35, P < 0.001), whereas the agreement between plasma phylloquinone and PIVKA-II concentrations was not as strong (r = -0.15, P < 0.05).

Assessment of dietary phylloquinone intake and vitamin K status in postmenopausal women.

OBJECTIVE: To examine the relationship between dietary phylloquinone intake and vitamin K status of postmenopausal Caucasian women. DESIGN: Cross-sectional study, in which dietary intake was estimated using weighed record techniques and vitamin K status was measured by a single plasma phylloquinone concentration and 24-h urinary gamma-carboxyglutamic acid (Gla) excretion. SETTING: The metabolic research unit at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA. SUBJECTS: 402 healthy postmenopausal Caucasian women who were participating in a randomized trial to determine the effect of calcium supplementation on bone loss. Of the original group, 362 had complete weighed diet records, 358 had corresponding plasma phylloquinone concentrations, and 346 had corresponding urinary Gla measurements. RESULTS: There was a significant correlation (r = 0.13, P = 0.01) between total dietary intake of phylloquinone (geometric mean = 89 micrograms/day) and plasma phylloquinone levels (mean = 1.12 nmol/l). Dietary intake was neither correlated with urinary Gla excretion (mean = 4.0 mumol/mmol creatinine) nor did it vary by season. The ratio of intra- to interindividual variance in phylloquinone intake was 2.6, from which it was estimated that 5 days of independent recording is necessary to estimate true usual dietary intake, assuming a correlation of 0.8. CONCLUSIONS: A weighed record has the potential to be a reliable method for estimating dietary intakes of vitamin K which relate to plasma phylloquinone levels used as an indicator of vitamin K status in postmenopausal Caucasian women.

Urinary levels of gamma-carboxyglutamic acid and its clinical significance.

Urinary gamma-carboxyglutamic acid (gamma-Gla) levels were determined in healthy subjects of all ages. The urinary gamma-Gla levels were highest in infants (0-1 years), then fell in an age-dependent manner, again in subjects reaching a minimum value in adults, then gradually increased over 60 years of age. Urinary gamma-Gla levels therefore change markedly with aging. The relationships between the urinary gamma-Gla excretion and plasma levels of prothrombin and protein C in patients with various hepatic diseases or diabetes mellitus were examined and compared with those in healthy adults. Both plasma prothrombin and protein C levels were decreased in all patients with liver disease compared with healthy adults. In patients with hepatitis and liver cirrhosis, the decrease did not, however, affect the gamma-Gla excretion. In addition, in patients with hepatoma or carcinoma with liver metastases, the urinary gamma-Gla levels were increased. In patients with diabetes mellitus, the urinary gamma-Gla levels and plasma levels of prothrombin and protein C tended to increase, but this was not significant. The present results indicate that simultaneous measurement of the levels of urinary gamma-Gla and plasma prothrombin and protein C is a useful tool for the diagnosis of liver diseases and diabetes mellitus.

Dietary induced subclinical vitamin K deficiency in normal human subjects.

A subclinical vitamin K deficiency was induced in 32 healthy subjects (four groups of eight males and females) aged 20-40 and 60-80 yr residing in the Metabolic Research Unit of the Human Nutrition Research Center on Aging at Tufts University. Volunteers were initially fed (4 d) a baseline-period diet containing the recommended daily allowance for vitamin K which is equivalent to 80 micrograms/d of phylloquinone (vitamin K1). During the baseline period various parameters of vitamin K nutritional status were monitored. The baseline period was followed by a 13-d depletion period during which the subjects were fed a very low vitamin K1 diet (approximately 10 micrograms/d). After depletion, the subjects entered a 16-d repletion period (four stages lasting 4 d each) during which time they were repleted with 5, 15, 25, and 45 micrograms of vitamin K1 per day. Vitamin K1 depletion dramatically and significantly decreased plasma vitamin K1 levels (P < 0.0001) in both elderly and young groups to values 13-18% of day 1 (elderly 0.22 nM, young 0.14 nM). Repleting the subjects with up to 45 micrograms of vitamin K1 per day failed, in the case of the young subjects, to bring plasma vitamin K1 levels back into the normal range. Dietary vitamin K1 restriction induced different responses in the urinary excretion of gamma-carboxyglutamic acid between the young and the elderly subjects with values decreasing significantly (P < 0.03) in the young while remaining unchanged in the elderly. The vitamin K1 depletion period had no significant effect on either prothrombin and activated partial thromboplastin times, or Factor VII and protein C (as determined by antigenic and functional assays). By using a monoclonal antibody, decarboxy prothrombin was found to increase slightly but significantly in both groups (P < 0.05) as a consequence of the low vitamin K1 diet. This study clearly shows that a diet low in vitamin K1 can result in a functional subclinical deficiency of vitamin K (decreased urinary gamma-carboxyglutamic acid excretion) without affecting blood coagulation.

[A study on urinary free gamma-carboxyglutamic acid in patients with idiopathic urinary calcium stone].

Currently, urinary excretion of free gamma-carboxyglutamic acid (gamma-gla.), a terminal amino acid degraded from gamma-gla. containing protein including bone Gla. Estimated to be a more specific marker for bone metabolism and useful clinically rather than urinary excretion of hydroxyproline. In addition, serum levels of BGP have proved to be a significantly valuable indicator for bone metabolism, especially for process of bone formation, in recent studies. Therefore, we measured these parameters in 40 patients with idiopathic urinary calcium (Ca) stone and investigated bone metabolism in those patients. However, in majority of cases studied, urinary levels of gamma-gla. as well as that of hydroxyproline proved to be definite difference from that in healthy subjects (n = 12) and failed to suggest the presence of abnormality in bone turnover in the background of stone formation. Urinary excretion of hydroxyproline were 6.68 +/- 3.89 micrograms/mg.Cr in the patients and 6.95 +/- 3.08 micrograms/mg.Cr in healthy subjects. Urinary excretion of gamma-gla were 55.0 +/- 15.8 nmol/mg.Cr in the patients and 47.2 +/- 7.3 nmol/mg.Cr in healthy subjects.

Determination of protein-bound urinary gamma-carboxyglutamic acid in calcium nephrolithiasis.

Nephrocalcin is a urinary gamma-carboxyglutamic acid (Gla) containing protein that may be a physiological inhibitor of calcium oxalate nephrolithiasis. Nephrocalcin isolated from urine of stone formers seems to be abnormal in lacking Gla that is required for inhibitory activity. In order to study this hypothesis, we compared the protein-bound urinary Gla contents in 32 calcium oxalate stone formers and in 24 controls. Protein-bound Gla was resolved by reversed-phase high-performance liquid chromatography after elimination of free Gla, alkaline hydrolysis and precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Protein-bound urinary Gla concentrations were similar in stone formers (0.83 +/- 0.38 mumol/l, mean +/- SD) and controls (0.81 +/- 0.27) and were less than 5% of free urinary Gla. However, excretion rates of free and protein-bound Gla (nmol/min) were higher in stone formers (P = 0.006 and P = 0.002). Positive correlations (P = 0.000) were observed between free and protein-bound Gla both in controls and in stone formers. These results do not support the hypothesis of a lacking Gla nephrocalcin in stone formers.

Use of non-collagen markers in osteoporosis studies.

Clinical and research laboratories routinely measure various hormonal and nonhormonal parameters of calcium and phosphorus metabolism, and markers of bone turnover. Such measurements may help clinical decision-making relating to metabolic bone disease and osteoporosis. Molecular biological and cell-culture techniques are being used in basic biochemical research on bone-cell metabolism. Results may aid understanding of normal and abnormal regulation of the bone-cell metabolism, and thus provide further insights relating to the diagnosis and prevention of osteoporosis.

Effects of aztreonam on fecal flora and on vitamin K metabolism.

The effects of aztreonam on fecal flora and on descarboxy prothrombin (PIVKA-II) in plasma and gamma-carboxyglutamic acid (Gla) in urine as an index of vitamin K metabolism were studied in seven children (age range, 2 months to 2 years) with urinary tract infections. Daily doses of aztreonam were 60 to 80 mg/kg. Stool specimens were obtained before the treatment, on days 3 to 5 of aztreonam use, and from 3 to 5 days after the cessation of treatment. The counts of enterobacteria decreased (P less than 0.01) and those of streptococci increased (P less than 0.05) during aztreonam treatment. The anaerobic organisms, especially bifidobacteria and bacteroides, showed no marked change. PIVKA-II and Gla were investigated before and during the treatment with aztreonam. PIVKA-II was not detected in seven patients before or during aztreonam use. There were no significant differences in the levels of Gla in urine before or during the treatment.