Noninvasive in vivo quantitative assessment of fat content in human liver.

BACKGROUND/AIMS: Since the introduction of ultrasonography, liver steatosis has become an increasingly frequent diagnosis. Both ultrasonography (US) and computerized tomography (CT) provide qualitative rather than quantitative assessment of fatty infiltration. The objective of this study was to develop a noninvasive method for the quantification of the hepatic fat content in vivo. A test object containing solutions with CT scan density (CTD) similar to normal liver ("liver-equivalent") or "fat-equivalent material" in variable proportions was prepared to measure patients with variable degrees of steatosis in vivo. RESULTS: A linear correlation (r=0.99, p<0.001) linked CTD and the increasing percentage of fat-equivalent material. A CTD calibration curve was derived as a reference for the in vivo determinations. In 29 consecutive patients with steatosis diagnosed by histology, CTD was linearly correlated (r=0.83, p<0.001) with the hepatic fat content (HFC) expressed as percent of the whole liver, obtained by a computerized histomorphometric analysis. Based on the calibration curve obtained in 29 subjects who underwent liver biopsy, 38 additional consecutive steatotic patients were examined and the degree of hepatic fat content was calculated. The HFC was linearly correlated (r=-0.86, p<0.001) with the liver-to-spleen ratio. CONCLUSIONS: We conclude that the use of test objects allows an accurate and reproducible noninvasive quantitative assessment of hepatic fat infiltration in humans. This technique may prove useful in the evaluation of the natural course and treatment of hepatic steatosis as well as in the assessment of donor livers prior to transplantation.

Proton MR spectroscopy in quantitative in vivo determination of fat content in human liver steatosis.

To demonstrate that the lipid volume fraction in liver steatosis can be accurately estimated with in vivo hydrogen-1 magnetic resonance (MR) spectroscopy, the authors developed a calibration procedure based on in vitro MR spectroscopy of lipid extracts from steatotic liver specimens. The lipid volume fractions determined with the calibration procedure were compared with the results of histomorphometry and with calibrated computed tomographic (CT) data. The volume fraction of fat determined with MR spectroscopy was in good agreement with the CT results, whereas histomorphometry underestimated the amount of hepatic fat. The results indicate that determination of the fat volume fraction in steatotic liver can be achieved noninvasively with MR spectroscopy.

The glycosides of hydroxylysine are final products of collagen degradation in humans.

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are metabolites derived from collagen degradation. They are useful biochemical markers provided they are not further processed. Experiments were run to test the activity of alpha- and beta-glycosidases in human kidney cortex preparations. Results allow to exclude the presence of the specific enzymes, in contrast with what is reported for the rat kidney tissue.

Transformation of fetal secondary cartilage into embryonic bone in organ cultures of human mandibular condyles.

Mandibular condyles of human fetuses, 14-21 weeks in utero, were kept in an organ culture system for up to 60 days. After 6 days in culture, the cartilage of the mandibular condyle appeared to have maintained its inherent structural characteristics, including all its various layers: chondroprogenitor, chondroblastic, and hypertrophic. After 12 days in culture, no chondroblasts could be seen; instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells in the vicinity of the chondroprogenitor zone differentiated into osteoblast-like cells that produced type I collagen. The progenitor cells were still actively incorporating 3H-thymidine. The newly formed osteoid-like tissue lacked both metachromatic reactivity and a response to antibodies against chondroitin sulfate. Instead, the tissue reacted positively for osteocalcin (bone gla-protein). The process of new bone formation further progressed and, by the 20th day in culture, the new bone reacted positively for type I collagen, osteonectin, and to a lesser extent for chondroitin sulfate. The osteoid also underwent mineralization as revealed by both the von Kóssa stain and vital staining with tetracycline. The above feature appeared even more intense in 40-day-old cultures. After 60 days, the newly formed bone contained osteoblasts and osteocytes, whereas the extracellular matrix revealed a high degree of matrix polarization. The results of the present study recapitulate findings reported for organ cultures of mice mandibular condyles. However, the in vitro process of de novo bone formation in human specimens requires a 6-fold longer culture time than that needed for mice condyles.

Biochemical markers for detecting bone metastases in patients with breast cancer.

A study was carried out to assess the best use of biochemical bone markers to exclude metastases in patients with breast cancer. Urinary galactosyl-hydroxylysine and serum alkaline phosphatase were used to monitor bone resorption and deposition, respectively. Hydroxyproline was also measured. In a selected population of patients, possibly affected by metastases on the basis of scintigraphic examination, which is highly sensitive but poorly specific, we assessed the efficiency of the markers by a double statistical analysis. In this group, the only marker able to predict metastases was galactosyl-hydroxylysine.

A new mathematical model to study bone turnover in growing rats.

A new mathematical model for the study of bone turnover in growing rats was developed. The model predicts a linear relationship between bone mineral content (BMC) and biochemical markers (BMK) of bone turnover assuming that rats are growing, bone turnover is profoundly affected by skeletal maturation, and resorption and formation are physiologically balanced. The model validation was performed by measuring galactosyl-hydroxylysine (GHYL) and hydroxyproline (HYP) in urines. This mathematical evidence supports our proposed use of the specific bone resorption marker GHYL to predict bone mineral content. Further studies on bone turnover will be possible by the application of the same approach.

Energy state of chondrocytes assessed by 31P-NMR studies of preosseous cartilage.

The energy state of resting and hypertrophic chondrocytes from growth plate was studied by 31P-NMR spectroscopy of superfused cartilage slices. The presence of phosphocreatine was demonstrated in both cell types, using a repetition time of 3 s. By comparing the decline in the nucleoside triphosphate level after adding blockers of the glycolysis or of the mitochondrial respiration, it was deduced that resting and hypertrophic chondrocytes use both metabolic pathways for energy production, but the glycolysis dominates. Hypertrophic cells rely more on the mitochondrial respiration than the resting cells.

Immunohistochemical studies of the extracellular matrix in the condylar cartilage of the human fetal mandible: collagens and noncollagenous proteins.

This study provides data concerning the cells and their extracellular matrix in prenatal human mandibular condylar cartilage. The latter cartilage represents a secondary type of cartilage since it develops late in the morphogenesis of the craniofacial skeleton. The cartilage of the mandibular condyle is actively involved in endochondral ossification, thus showing all the phases of cartilage growth, maturation, and mineralization that precedes de novo bone formation. The present study focused on the localization and distribution of the major macromolecules that are normally encountered in cartilage and bone, including collagens, proteoglycans, fibronectin, osteonectin, osteocalcin, alkaline phosphatase, and anchorin CII. It became clear that the mineralized zone of the cartilage already contained bone-specific antigens; thus the above zone might serve as an essential propagative predecessor in the ossification process.

Serum-induced cytosolic calcium movements and mitogenesis in cultured preosseous chondrocytes.

The differentiation of preosseous chondrocytes begins with the proliferation of resting cells and results in the expression of the hypertrophic phenotype. The effect of fetal calf serum on chondrocyte mitogenesis and intracellular Ca2+ concentration was studied in resting and hypertrophic cells in primary culture. Resting chondrocytes respond to the growth stimulus with immediate release of Ca2+ from intracellular stores and with opening of the plasma membrane Ca2+ channels. These events may be related to the elevated [3H]thymidine incorporation observed after serum exposure. In contrast, in hypertrophic chondrocytes the lower rate of DNA synthesis seems to be coupled with a lower activity of the Ca2+ signaling mechanism and, probably, with reduced intracellular calcium stores. It is proposed that expression of the Ca2+ signaling mechanism may be modulated during the differentiation of preosseous chondrocytes.

Evidence in vitro for an enzymatic synthesis of phosphocitrate.

A biological synthesis of phosphocitrate is described from precursors, citrate and adenosinetriphosphate reacting in the presence of rat liver homogenate. Identity of the newly formed product was examined by enzymatic digestion of reactions mixtures, HPLC chromatography and 1H-NMR spectra. Authenticity of product was established by comparison to chemically synthesized phosphocitrate. Recognition of the existence of a biologically synthetic pathway adds credence to the known presence of phosphocitrate in mitochondria and a postulated role to control calcium phosphate deposition in that organelle.

Modification of plasma membrane of differentiating preosseous chondrocytes: evidence for a degradative process in the mechanism of matrix vesicle formation.

Chondrocytes of the growth plate are differentiating cells. Their evolution leads to matrix vesicle formation and to cartilage mineralization. This is an in vitro study of the plasma membrane of chondrocytes at two differentiation stages. Differences in protein and glycoprotein components, increased membrane fluidity, and responsiveness to PTH indicate that hypertrophic ("ossifying") chondrocytes possess a plasma membrane widely different from that of resting chondrocytes. Their plasma membrane is particularly enriched in alkaline phosphatase (Mr 70K). Purified matrix vesicles contain the 70K form of alkaline phosphatase, but a 50K species is also detectable, a signal of degradative process. In fact, proteins and glycoproteins of matrix vesicles are less numerous than those of cell plasma membranes. It is suggested that, in vivo, matrix vesicle formation may be mediated by Ca2(+)-activated neutral proteases.

High predictivity of galactosyl-hydroxylysine in urine as an indicator of bone metastases from breast cancer.

We measured the urinary excretion of galactosyl-hydroxylysine (GH) and hydroxyproline in two groups of women with breast cancer, with (M+, n = 24) and without (Mo, n = 30) clinical, scintigraphic, or radiological evidence of bone metastases. Both these compounds are excreted in larger amounts in the M+ group than in the Mo patients. However, GH, which is a specific marker for bone collagen, provides better predictivity for bone metastases than does hydroxyproline: 92% sensitivity and 90% specificity vs 74% and 79%, respectively, for hydroxyproline.

Possible mechanism of inhibition of cartilage alkaline phosphatase by insulin.

The inhibition of alkaline phosphatase by insulin is a finding reported by many researchers but the mechanism of this inhibition has not been studied. Since alkaline phosphatase is an important factor in the mechanism of calcification and an impairment of mineralisation has been observed in diabetes mellitus, a study was carried out to assess the effect of the hormone on alkaline phosphatase measured in chondrocytes, in matrix vesicles and in a purified enzyme preparation. Enzyme activity was inhibited by insulin. The lowest active concentration was 10(-6) M and maximal inhibition was obtained at about 10(-4) M. The inhibition is of the uncompetitive type. Full recovery of the hormone-inhibited enzyme was obtained with 10(-4) M 2-mercaptoethanol. Data suggest direct interaction between the alkaline phosphatase and insulin molecules, involving either disulfide cross linkages or the metal chelating activity of insulin.

High-performance liquid chromatographic preparation of galactosyl-hydroxylysine, a specific bone collagen marker.

Galactosyl-hydroxylysine, a specific bone collagen marker, has been prepared directly from human urine samples by high-performance liquid chromatography (HPLC) on a preparative column. The compound is the didansyl derivative, as proved by HPLC and mass spectrometry under fast atom bombardment conditions. Since this compound is not commercially available, the procedure reported appears to be the simplest way to prepare it, which is necessary to measure the urinary excretion of this collagen metabolite by HPLC.

Urinary beta-1-galactosyl-0-hydroxylysine (GH) as a marker of collagen turnover of bone.

beta-1-galactosyl-0-hydroxylysine (GH) was measured in the urine of 59 women and 48 men, aged 30-79 years, by High Performance Liquid Chromatography (HPLC) of the dansylated derivative. Vertebral mineral density, measured by quantitative computed tomography (QCT), and urinary GH were inversely correlated (r = -0.74; P less than 0.001). High rate of bone mineral loss is associated with a high urinary GH excretion. Measurement of GH in urine provides a simple and noninvasive method for the evaluation of the extent of bone resorption in large groups of subjects and appears to be more specific than urinary hydroxyproline excretion.

Determination of galactosyl hydroxylysine in urine as a means for the identification of osteoporotic women.

A sensitive and specific method is proposed to follow bone collagen degradation. The procedure consists of the measurement of galactosyl hydroxylysine (GH) in urine by HPLC. The aim of the work is to assess the predictive values of the method for the identification of post-menopausal osteoporotic women. By assuming the value of 12 mumol/g creatinine as the threshold value, the sensitivity of the test is 87% and the specificity 60%. Individuals with a GH/creatinine ratio of 12 or below are not likely to be at risk of bone fractures: an equivalent predictive value is provided by the measurement of bone density by quantitative computed tomography. This biochemical method is however simple and not invasive and may be frequently repeated.

Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein.

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.

A possible role for polyamines in cartilage in the mechanism of calcification.

The role of polyamines in cartilage is not known: they may be somehow related to the mechanism of calcification. In epiphyseal cartilage from calf scapulas, they are more concentrated in the ossifying area, where calcification takes place, than in the resting region. Spermidine is present in greater amounts than spermine and putrescine. Since ornithine decarboxylase (EC 4.1.1.17) is measurable only in the resting region of the tissue, it is in this area that polyamine biosynthesis occurs, while they accumulate in the ossifying area. Immunohistochemical evidence is obtained that only in the ossifying zone is spermidine extracellular. It is at this level that the matrix is rearranged to become calcified, and proteoglycans are dissociated and partially removed. The effect of polyamines on solutions of proteoglycan subunits has been studied in vitro by following variations of turbidity and viscosity. While in the presence of putrescine the specific viscosity decreases to asymptotic values, in the presence of either 30 mM spermidine or 2.5-10 mM spermine, the decrement is more marked. At the same concentrations, increase of the turbidity of proteoglycan subunit solutions was observed. Only spermidine showed the capacity of displacing proteoglycan subunits from a column of Sepharose 4B-type II collagen: at 15 mM concentration, about 90% of proteoglycans were removed from the column. Alkaline phosphatase activity, which plays an important role in calcification, is enhanced by spermidine and spermine. These results obtained in vitro support the hypothesis that polyamines may be related to calcification of preosseous cartilage.