Predictors of CNS injury as measured by proton magnetic resonance spectroscopy in the setting of chronic HIV infection and CART.

The reasons for persistent brain dysfunction in chronically HIV-infected persons on stable combined antiretroviral therapies (CART) remain unclear. Host and viral factors along with their interactions were examined in 260 HIV-infected subjects who underwent magnetic resonance spectroscopy (MRS). Metabolite concentrations (NAA/Cr, Cho/Cr, MI/Cr, and Glx/Cr) were measured in the basal ganglia, the frontal white matter, and gray matter, and the best predictive models were selected using a bootstrap-enhanced Akaike information criterion (AIC). Depending on the metabolite and brain region, age, race, HIV RNA concentration, ADC stage, duration of HIV infection, nadir CD4, and/or their interactions were predictive of metabolite concentrations, particularly the basal ganglia NAA/Cr and the mid-frontal NAA/Cr and Glx/Cr, whereas current CD4 and the CPE index rarely or did not predict these changes. These results show for the first time that host and viral factors related to both current and past HIV status contribute to persisting cerebral metabolite abnormalities and provide a framework for further understanding neurological injury in the setting of chronic and stable disease.

Asian women have greater abdominal and visceral adiposity than Caucasian women with similar body mass index.

BACKGROUND: In the Multiethnic Cohort Study, Japanese Americans (JA) have lower mean body mass index (BMI) compared with Caucasians, but show a higher waist-to-hip ratio at similar BMI values and a greater risk of diabetes and obesity-associated cancers. OBJECTIVE: We investigated the abdominal, visceral and hepatic fat distribution in these Asian and Caucasian Americans. DESIGN: A cross-sectional sample of 60 female cohort participants (30 JA and 30 Caucasians), of ages 60-65 years and BMIs 18.5-40 kg m(-2), underwent anthropometric measurements and a whole-body dual energy X-ray absorptiometry (DXA) scan: a subset of 48 women also had abdominal magnetic resonance imaging (MRI). RESULTS: By design, JA women had similar BMIs (mean 26.5 kg m(-2)) to Caucasian women (27.1 kg m(-2)). JA women were found to have a significantly smaller hip circumference (96.9 vs 103.6 cm; P=0.007) but not a significantly lower DXA total fat mass (25.5 vs 28.8 kg; P=0.16). After adjusting for age and DXA total fat mass, JA women had a greater waist-to-hip ratio (0.97 vs 0.89; P<0.0001), DXA trunk fat (15.4 vs 13.9 kg; P=0.0004) and MRI % abdominal visceral fat (23.9 vs 18.5%; P=0.01) and a lower DXA leg fat mass (8.2 vs 10.0 kg; P=<.0001). Their MRI % subcutaneous fat (33.4 vs 30.2%; P=0.21) and % liver fat (5.8 vs 3.8%; P=0.06) did not significantly differ from that of Caucasian women. CONCLUSIONS: Our findings build on limited past evidence, suggesting that Asian women carry greater abdominal and visceral fat when compared with Caucasian women with similar overall adiposity. This may contribute to their elevated metabolic risk for obesity-related diseases.

Altered neurometabolites and motor integration in children exposed to methamphetamine in utero.

Methamphetamine (METH) is a neurotoxic drug. This study aimed to evaluate brain metabolite levels and cognitive function in young children with prenatal METH exposure. 101 children ages 3-4 years were evaluated with neuropsychological tests and underwent proton magnetic resonance spectroscopy ((1)H-MRS) without sedation. Complete datasets from 49 METH-exposed and 49 controls who completed the neuropsychological test battery, and 38 METH-exposed and 37 controls with high-quality MR spectra are reported here. Despite similar physical characteristics (including head circumference), global cognitive function (on Stanford-Binet), parental education, intelligence, mood, and socioeconomic status, METH-exposed children had higher total creatine (tCr: +7%, p=0.003), N-acetyl compounds (NA: +4.3%, p=0.004) and glutamate+glutamine (GLX: +9.6%, p=0.02) concentrations in the frontal white matter, but lower myoinositol (MI: -7%, p=0.01) and MI/tCr (-7.5%, p=0.03) in the thalamus, than control children. The higher frontal white matter NA in the METH-exposed children was due to the higher NA in the METH-exposed girls (+10.2%, p=0.003), but not the boys (+0.8%) compared to sex-matched controls. Furthermore, the METH-exposed children had poorer performance on a visual motor integration (VMI) task, which correlated with lower MI in the thalamus (r=0.26, p=0.03). The higher NA, tCr and GLX concentrations suggest higher neuronal density or cellular compactness in the white matter, especially in the girls, whereas the lower MI suggests lower glial content in the thalamus of these METH-expose children. These findings combined with their poorer performance on VMI also suggest accelerated but aberrant neuronal and glial development in these brain regions.

Abnormal myocardial phosphorus-31 nuclear magnetic resonance spectroscopy in women with chest pain but normal coronary angiograms.

BACKGROUND: After hospitalization for chest pain, women are more likely than men to have normal coronary-artery angiograms. In such women, myocardial ischemia in the absence of clinically significant coronary-artery obstruction has long been suspected. Most methods for the detection of the metabolic effects of myocardial ischemia are highly invasive. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy is a noninvasive technique that can directly measure high-energy phosphates in the myocardium and identify metabolic evidence of ischemia. METHODS: We enrolled 35 women who were hospitalized for chest pain but who had no angiographically significant coronary-artery obstructions and 12 age- and weight-matched control women with no evidence of heart disease. Myocardial high-energy phosphates were measured with 31P-NMR spectroscopy at 1.5 tesla before, during, and after isometric handgrip exercise at a level that was 30 percent of the maximal voluntary grip strength. We measured the change in the ratio of phosphocreatine to ATP during exercise. RESULTS: Seven (20 percent) of the 35 women with chest pain and no angiographically significant stenosis had decreases in the phosphocreatine:ATP ratio during exercise that were more than 2 SD below the mean value in the control subjects without chest pain. There were no significant differences between the two groups with respect to hemodynamic variables at rest and during exercise, risk factors for ischemic heart disease, findings on magnetic resonance imaging and radionuclide perfusion studies of the heart, or changes in brachial flow during the infusion of acetylcholine. CONCLUSIONS: Our results provide direct evidence of an abnormal metabolic response to handgrip exercise in at least some women with chest pain consistent with the occurrence of myocardial ischemia but no angiographically significant coronary stenoses. The most likely cause is microvascular coronary artery disease.

31P-magnetic resonance spectroscopy studies of cardiac transplant patients at rest.

Studies in animal models and patients have suggested that 31P-magnetic resonance spectroscopy (MRS) may be useful in diagnosing transplant rejection, but such studies often are confounded by the late inclusion of patients after transplantation. The present study examined the utility of 31P-MRS in the diagnosis of acute allograft rejection during the first posttransplant month. Thirteen recent heart transplant recipients underwent 57 resting 31P-MRS studies within 24 hr of a biopsy. Subjects lay supine with a 10-cm surface coil placed over the heart. A 1-dimensionsal chemical shift imaging protocol was used to collect spectral information. Spectra from the heart were weighted for distance from the coil and summed before analysis. ANOVA and Duncan's multiple range test were used to analyze the data comparing phosphocreatine (PCr)/ATP ratios with biopsy scores. Transplant patients had significantly lower myocardial PCr/ATP ratios when compared with a normal control group (1.27 +/- 0.27 versus 1.61 +/- 0.22, p < 0.001). However, when the patient group was classified by biopsy score, the expected order of score, 0 > 1 > 2 > 3, was not obtained. Rather, the order was 2 > 0 > 1 > 3. Although the difference between scores 2 and 3 was significant (1.46 versus 1.14, alpha = 0.05 level), the lower three groups were statistically indistinguishable. In addition, the PCr/ ATP ratios were not predictive of future biopsies. Although significantly lower than normal control subjects, resting myocardial PCr/ATP ratios of transplant subjects are not useful in assessing thelevel of rejection. It is suggested that the measurement may be more predictive in mildly exercised myocardium.

Brain lesions in patients with AIDS: H-1 MR spectroscopy.

PURPOSE: To evaluate the role of proton (hydrogen-1) magnetic resonance (MR) spectroscopy in the differential diagnosis of focal brain lesions in patients with acquired immunodeficiency syndrome (AIDS). MATERIALS AND METHODS: Twenty-six men with 35 AIDS-related brain lesions underwent MR imaging and localized H-1 MR spectroscopy. Lesions consisted of 11 toxoplasmic abscesses, 12 progressive multifocal leukoencephalopathic lesions, eight lymphomas, and four cryptococcomas. Metabolite peak areas in the lesions were compared with those in the contralateral hemisphere in each patient. RESULTS: H-1 MR spectroscopic findings showed significantly different biochemical profiles for each diagnostic group (P = .0001) with regard to N-acetyl compounds, total creatine pool, choline-containing compounds, myoinositol, and lactate. H-1 MR spectroscopy alone helped correctly diagnose 94% (84% with jackknifed classification) of the brain lesions, without overlap between toxoplasmosis and lymphoma. CONCLUSION: H-1 MR spectroscopy is a sensitive and potentially specific noninvasive adjunctive method for differential diagnosis of focal brain lesions in AIDS.

Analysis of brain tumors using 1H magnetic resonance spectroscopy.

BACKGROUND: Magnetic resonance imaging (MRI) is superior in delineating anatomic and pathologic information and has subsequently been married to the ability of magnetic resonance spectroscopy (MRS) to provide insight into the biochemical changes underlying pathology. Proton magnetic resonance spectroscopy (1H MRS) allows the non-invasive in vivo collection and measurement of chemical information from a selected volume of tissue (voxel). METHODS: We conducted a prospective trial in 23 patients with brain mass lesions and 16 normal subjects using proton magnetic resonance spectroscopy (1H MRS). The spectra were analyzed for N-acetyl-aspartate (NAA), choline compounds (Cho), creatine (Cr), and lactate (Lac). The ratios of the compounds in tumors were compared to normals. RESULTS: The tumors showed significant decreases in the mean peak height ratios of NAA/Cho, NAA/Cr, and significant increases in Cho/Cr when compared to tissue from normal subjects. Cho was elevated in all of the meningiomas and gliomas. In benign tumors, Cho was usually elevated while in metastases Cho was often normal or decreased. The four metastatic tumors showed NAA/Cho, NAA/Cr, and Cho/Cr that were similar to controls. Lac varied with tumor type and was elevated in many malignant primary brain tumors. CONCLUSIONS: 1H MRS is a powerful tool for safe, noninvasive analysis of tissue chemistry in vivo. Analysis of intracranial tumors reveals significant trends that might eventually be used in the classification of tumor histology and evaluation of the efficacy of tumor treatment.

Localized in vivo 1H magnetic resonance spectroscopy and in vitro analyses of heterogeneous brain tumors.

Results of magnetic resonance spectroscopic (MRS) studies of the chemical patterns in brain tumors have been inconsistent. Actual biochemical correlations are needed. In 2 patients with heterogeneous intracranial tumors, in vivo 1H MRS and in vitro biochemical analyses were correlated. Histology confirmed the tumor heterogeneity. Choline was elevated in the cellular portion of both tumors but decreased in the necrotic or cystic portions. Creatine was diffusely decreased while lactate was elevated in all regions of both tumors. Furthermore, the increase in the choline peak on 1H MRS appeared to be due to increases in water-soluble choline compounds. This study illustrates the value of small localized voxels for differentiating regional chemical differences in tumors.

Changes in potential controllers of human skeletal muscle respiration during incremental calf exercise.

The purpose of this study was to evaluate the consequences of non-linear changes in phosphocreatine (PCr) and pH during incremental calf exercise on estimates of ADP and cytosolic free energy of ATP hydrolysis (delta GATP). Six subjects performed incremental plantar flexion exercise on a treadle ergometer while muscle P(i) metabolism (PCr, P(i), ATP) and pH were followed using 31P-nuclear magnetic resonance spectroscopy. Changes in ADP and delta GATP were estimated with the assumption that there was equilibrium of the creatine kinase reaction and homogeneous tissue metabolite pools. All six subjects showed a threshold for onset of cellular acidosis that occurred on average at 47.3 +/- 12.7% of peak work rate (PWR). In five of the six subjects, PCr and P(i) showed accelerated rates of change above the threshold for onset of cellular acidosis. In all six subjects, ADP, when correctly calculated considering changes in pH, rose in a curvilinear fashion that was well described by a Michaelis-Menten hyperbola through 60-100% of PWR, with a mean apparent Michaelis-Menten constant of 43.1 +/- 17.1 microM ADP and a predicted maximal oxidative rate at PCr = 0, which was 241 +/- 94% of PWR. delta GATP rose linearly with work rate from -62.9 +/- 1.8 kJ/mol during unloaded treadling to -55.0 +/- 1.8 kJ/mol at PWR. If we assume a linear O2 uptake-to-work rate relationship, these results are most consistent with control of respiration being exerted through delta GATP under these conditions (incremental exercise by human calf muscle).(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle energetics and pulmonary oxygen uptake kinetics during moderate exercise.

The present study tested whether, during moderate exercise, 1) the dynamic responses of ADP and changes in free energy of ATP hydrolysis (delta GATP) were similar to those of phosphocreatine [PCr; as would be expected for a simple controller of muscle respiration (QO2)] and 2) the rise in pulmonary O2 uptake (VO2) during cycle exercise would reflect the rise in muscle QO2 indicated by the calf PCr kinetics. The responses of PCr, Pi, ADP, and delta GATP were measured from the calf in five subjects during supine treadle exercise using 31P-magnetic resonance spectroscopy and compared with those for VO2, measured breath by breath during upright cycle exercise. The time constants for delta GATP [24.2 +/- 14.2 (SE) s] were not significantly different from those for PCr (26.3 +/- 17.3 s) and Pi (30.7 +/- 22.5 s) (P > 0.05). The time constants for phase 2 VO2 (29.9 +/- 16.8 s) were also similar to those of PCr. In contrast, the dynamics of ADP were distorted from those of PCr due to dynamic changes in pH. These results are consistent with mechanisms of respiratory control that feature substrate control by PCr or thermodynamic control through changes in delta GATP. However, these results are not consistent with substrate control by ADP in a simple fashion. Furthermore, the similarity of time constants for phase 2 VO2 and muscle PCr suggests that phase 2 VO2 kinetics reflect those of muscle QO2 in healthy subjects during moderate exercise.

In vivo proton spectroscopy of intracranial infections and neoplasms.

The chemical characteristics of 10 neoplastic and 11 infectious brain masses were studied by in vivo 1H magnetic resonance spectroscopy. In tumors, peak height ratios of n-acetyl-L-aspartate to choline were decreased compared to those in normal brain tissue and infectious masses (p < 0.02), but the ratios in normal brains and those with infections did not differ. N-acetyl-L-aspartate-to-creatine/phosphocreatine ratios were significantly lower in infectious masses and tumors compared to normal brain tissue (p = 0.003). However, in progressive multifocal leukoencephalopathy, N-acetyl-L-aspartate appeared relatively unchanged. Lactate was greater than choline in 9 of 11 brains with infection, 0 of 14 control brains, and 1 of 10 tumors. Lactate-to-choline ratios were significantly elevated in infectious masses compared with tumors (p < 0.01). 1H magnetic resonance spectroscopy is promising for the noninvasive diagnosis of focal brain masses.

31P-magnetic resonance spectroscopy of leg muscle metabolism during exercise in children and adults.

Gas exchange response to high-intensity exercise differs in children and adults. We hypothesized that these findings are related to a lower anaerobic ATP supply in children. We predicted therefore a maturation of muscle high-energy phosphate metabolism during exercise. To test this hypothesis, we measured calf muscle Pi, phosphocreatine (PCr), and pH with 31P-nuclear magnetic resonance spectroscopy during rest and progressive exercise in 10 children and 8 adults. No differences were found at rest in pH and Pi/PCr between children and adults. Exercise resulted in a greater increase in Pi/PCr (P < 0.001) and decrease in pH (P < 0.0001) in adults than in children. Six adults and five children exhibited a transition from a slow to a faster rate of Pi/PCr increase and pH decrease during exercise. No significant differences were found between the two groups in the initial slow-phase slopes of Pi/PCr and pH as a function of work rate. In contrast, during the fast phase, Pi/PCr increased (slope: adults 23.6 +/- 9.8, children 10.7 +/- 2.5; P < 0.05) and pH decreased (slope: adults -6.0 +/- 1.9, children -3.7 +/- 1.2; P < 0.05) more rapidly in adults than in children. In conclusion, high-intensity exercise results in different kinetics of Pi/PCr and pH between children and adults. These results suggest that children are less able than adults to affect ATP rephosphorylation by anaerobic metabolic pathways during high-intensity exercise.

Hepatic D-galactosamine toxicity studied with localized in vivo 31P magnetic resonance spectroscopy in intact rats.

Spatially resolved 31P magnetic resonance spectroscopy (MRS) at 4.7 T was applied to noninvasively assess liver phosphorus metabolites in a biochemically well-characterized model of hepatotoxicity induced by injection of a sublethal dose of D-galactosamine (galN). A newly developed hybrid method based on spectral localization with B0 and B1 gradients was employed to obtain multivoxel spectra in intact anesthesized rats. Spatially localized in vivo spectra were recorded 0 to 26 h after galN injection of female rats. In response to galN exposure, diphosphodiester peaks ascribed to UDP-hexosamines became detectable by 4 h and persisted up to 26 h. A metabolite coresonating with inorganic phosphate increased rapidly in intensity by 2 h after galN and returned to baseline by 18 h; this resonance was shown not to be Pi and was assigned to galN-1-phosphate by subsequent high resolution MRS experiments on extracts prepared from these livers. These results confirmed in vivo the metabolic perturbations described previously for this model of hepatotoxicity following biochemical studies based on classical extraction methods. Unlike the in vitro studies, however, these noninvasive experiments provided additional information on the time course of metabolic alterations on the same animal.

In vivo T1 values of phosphorus metabolites in human liver and muscle determined at 1.5 T by chemical shift imaging.

A procedure for obtaining T1 values for phosphorous metabolites in localized regions of human subjects, using a standard 1.5 T MR imager, is described. 31P spectra and T1 values localized to the liver, and to abdominal and calf muscle of healthy volunteers were obtained by means of a multi-slice spectroscopy technique, consisting of a chemical shift imaging (CSI) sequence with a B1-insensitive excitation and one dimension of phase encoding, used in conjunction with a surface coil. An examination consisting of proton imaging, shimming and collection of 31P progressive saturation spectroscopic data for T1 determination required 1 h to perform. Shimming on the signal from the body region detected by the surface coil gave spectra of excellent spectral resolution. Quantification of all peaks in the localized 31P spectra was carried out with the PIQABLE algorithm, and T1 values were calculated for inorganic phosphate (Pi), the phosphodiester region, and the ATP alpha-, beta- and gamma peaks of liver, and for calf muscle Pi, phosphocreatine (PCr), and the three ATP peaks. The precisions of the measurement and of the entire process for obtaining and quantifying localized spectra by one-dimensional CSI were determined, and the accuracy of T1 values obtained by this means was verified. The temporal variation in T1 values obtained in a series of examinations of a single normal subject was also assessed. The consistency of the T1 values obtained in this study with in vivo T1 values obtained by other techniques is a stringent test of accuracy of localized spectra obtained with CSI.

Assessing the value of identifying the presence of malignant disease in human plasma by proton nuclear magnetic resonance spectroscopy.

PURPOSE: This study was performed to assess the ability of the average linewidths of the methyl and methylene resonances from the proton nuclear magnetic resonance (NMR) spectrum of human plasma to distinguish reliably between a normal, apparently healthy population and untreated patients with cancer as was suggested by a recent report (Fossel et al, N Engl J Med 1986; 315: 1369-1376). In that report, the absence of overlap between the two populations suggested that the technique had great diagnostic potential. SUBJECTS AND METHODS: Blood samples were collected from healthy hospital personnel and patients with biopsy proven cancer who had not yet undergone chemotherapy or radiotherapy. NMR measurements were obtained on decanted plasma samples. In addition, to determine whether the distributions obtained were sensitive to experimental conditions, we varied a number of experimental parameters. These were storage temperature, observation temperature, NMR observation technique, and magnetic field inhomogeneity. RESULTS: In our hands, the distributions substantially overlapped, although the means of the average linewidths of the normal (35.0 +/- 5.3 Hz, range = 24 to 48 Hz) and patient (31.6 +/- 5.7 Hz, range 20 to 44 Hz) populations were significantly different, delta = 3.4 Hz, p = 0.023. The degree of overlap corresponded to 74 percent sensitivity and 59 percent specificity. The distributions were independent of the experimental conditions except for observation temperature. In this case, there was insufficient difference between our experimental conditions and those of Fossel et al (25 degrees C versus 22 degrees C) to explain the difference in results. CONCLUSION: Our findings suggest that wide variations exist among normal, apparently healthy persons and emphasize the need for carefully matched control subjects. We conclude that this measurement is, as of now, unsuitable as a method for identifying malignant disease.

Alpha 1-adrenergic receptors in the adult, neonatal, and fetal canine heart.

Using the radioligand, iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([125I]-IBE 2254), alpha 1-adrenergic receptors were identified in membranes isolated from fetal, neonatal, and adult canine ventricular myocardium. Binding of the radioligand to alpha 1-adrenergic sites in adult canine heart was rapid, reversible, stereoselective and saturable. Computer analysis of binding data indicated 2 classes of receptors; one with very high affinity and limited capacity (Kd = 13 +/- 9 pM, Bmax = 25 +/- 15 fmol/mg, n = 5) and a second site with lower affinity and greater capacity (Kd = 1.20 +/- 0.43 nM, Bmax = 510 +/- 165 fmol/mg). Two sites were also identified in membranes isolated from fetal and neonatal canine ventricular myocardium. At the higher affinity site, all the age groups displayed similar capacities and affinities. At the lower affinity site, however both fetal and neonatal membranes displayed greater capacity than that of the adult.

Vitamin K-dependent processes in tumor cells.

Tumor cells are known to interfere with blood coagulation pathways of the host by producing procoagulants and other substances, thereby deriving certain advantages relating to tumor growth, metastasis, and angiogenesis. Anticoagulants may diminish these advantages under certain conditions. The interaction between coumarin anticoagulants and tumor cells has been reviewed with respect to procoagulants and their vitamin K-dependent properties. Evidence is also presented which suggests that vitamin K-dependent protein carboxylation is a general property of tumor cells.

Developmental changes in the effects of beta-adrenergic blocking concentrations of propranolol on canine Purkinje fibers.

We used standard microelectrode techniques to investigate the developmental changes in the effects of the beta-blocker propranolol on canine Purkinje fibers. The effects of propranolol on the transmembrane action potential were similar in neonatal and adult Purkinje fibers. In automatic Purkinje fibers we obtained concentration-response (increase in rate) curves for isoproterenol alone and with an increasing concentration of propranolol. The curve for isoproterenol in neonatal Purkinje fibers had an EC50 of 1.0 X 10(-9) M, about 5-fold lower than in adult Purkinje fibers (EC50 = 5.03 X 10(-9) M, p less than 0.05). At both ages, propranolol caused a parallel, concentration-dependent shift of the response curves for isoproterenol, with no change in the maximum effect. Equiactive dose ratios in presence and absence of 3 concentrations of propranolol were compared by the Schild plot. The pA2 values for adult and neonate Purkinje fibers were 8.32 (8.14-8.46; 95% confidence level) and 8.86 (8.69-9.03; 95% confidence level), respectively. Our results indicate significant (p less than 0.05) developmental changes in the beta-blocking effects of propranolol in canine Purkinje fibers.

Vitamin K dependent carboxylation of glutamate residues to gamma-carboxyglutamate in microsomes from spleen and testes: comparison with liver, lung, and kidney.

Vitamin K dependent carboxylation of glutamate residues to gamma-carboxyglutamate was demonstrated in proteins of spleen and testes microsomes. The rate of carboxylation in spleen microsomes was 0.9% and testes 3% of that in liver microsomes per milligram of microsomal protein. For comparison the rates of carboxylation in lung and kidney microsomes were 17 and 8%, respectively, of the rate in liver microsomes. The high rate in liver microsomes may be due to a high carboxylase level as indicated by the high rate of peptide carboxylation in liver microsomes. Protein carboxylation in liver microsomes was linear for only 15 min while carboxylation in microsomes from extrahepatic tissue persisted much longer so that the total protein carboxylation in lung microsomes was 60%, kidney 18%, testes 12%, and spleen 9% of that occurring in liver microsomes. Protein carboxylation was higher in microsomes from extrahepatic tissues of rats fed a vitamin K deficient diet as compared to animals fed a vitamin K sufficient diet. Protein carboxylation in microsomes from extrahepatic tissues was greatly stimulated by manganese ions and was dependent upon the addition of dithioerythritol. NADH could partially replace the dithiol in spleen, testes, and lung, but NADH-dependent carboxylation was relatively low in kidney and liver microsomes. Dithiol-dependent carboxylation was completely blocked by 10 microM warfarin, but NADH-dependent carboxylation was only slightly inhibited by 100 microM warfarin. Menaquinone-3 was much more active than vitamin K1 in driving carboxylation. Solubilized microsomes catalyzed the carboxylation of glutamate residues to gamma-carboxyglutamate in a pentapeptide Phe-Leu-Glu-Glu-Leu. The rate of carboxylation in lung microsomes was 22%, testes 3.3%, kidney 1.9%, and spleen 1.6% of the rate in liver microsomes.