The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a new, non-radioactive, endogenous labeling technique, we report direct measurements of circulating T-cell kinetics in normal and in HIV-1-infected humans. In healthy, HIV-1-seronegative subjects, CD4+ and CD8+ T cells had half-lives of 87 days and 77 days, respectively, with absolute production rates of 10 CD4+ T cells/microl per day and 6 CD8+ T cells/microl per day. In untreated HIV-1-infected subjects (with a mean CD4 level of 342 cells/microl), the half-life of each subpopulation was less than 1/3 as long as those of healthy, HIV-1-seronegative subjects but was not compensated by an increased absolute production rate of CD4+ T cells. After viral replication was suppressed by highly active antiretroviral therapy for 12 weeks, the production rates of circulating CD4+ and CD8+ T cells were considerably elevated; the kinetic basis of increased CD4 levels was greater production, not a longer half-life, of circulating cells. These direct measurements indicate that CD4+ T-cell lymphopenia is due to both a shortened survival time and a failure to increase the production of circulating CD4+ T cells. Our results focus attention on T-cell production systems in the pathogenesis of HIV-1 disease and the response to antiretroviral therapy.

Mass isotopomer distribution analysis (MIDA) is a technique for measuring the synthesis of biological polymers. First developed approximately eight years ago, MIDA has been used for measuring the synthesis of lipids, carbohydrates, and proteins. The technique involves quantifying by mass spectrometry the relative abundances of molecular species of a polymer differing only in mass (mass isotopomers), after introduction of a stable isotope-labeled precursor. The mass isotopomer pattern, or distribution, is analyzed according to a combinatorial probability model by comparing measured abundances to theoretical distributions predicted from the binomial or multinomial expansion. For combinatorial probabilities to be applicable, a labeled precursor must therefore combine with itself in the form of two or more repeating subunits. MIDA allows dilution in the monomeric (precursor) and polymeric (product) pools to be determined. Kinetic parameters can then be calculated (e.g., replacement rate of the polymer, fractional contribution from the endogenous biosynthetic pathway, absolute rate of biosynthesis). Several issues remain unresolved, however. We consider here the impact of various deviations from the simple combinatorial probability model of biosynthesis and describe the analytic requirements for successful use of MIDA. A formal mathematical algorithm is presented for generating tables and equations ( ), on the basis of which effects of various confounding factors are simulated. These include variations in natural isotope abundances, isotopic disequilibrium in the precursor pool, more than one biosynthetic precursor pool, incorrect values for number of subunits present, and concurrent measurement of turnover from exogenously labeled polymers. We describe a strategy for testing whether isotopic inhomogeneity (e.g., an isotopic gradient or separate biosynthetic sites) is present in the precursor pool by comparing higher-mass (multiply labeled) to lower-mass (single- and double-labeled) isotopomer patterns. Also, an algebraic correction is presented for calculating fractional synthesis when an incomplete ion spectrum is monitored, and an approach for assessing the sensitivity of biosynthetic parameters to measurement error is described. The different calculation algorithms published for MIDA are compared; all share a common model, use overlapping solutions to computational problems, and generate identical results. Finally, we discuss the major practical issue for using MIDA at present: quantitative inaccuracy of instruments. The nature and causes of analytic inaccuracy, strategies for evaluating instrument performance, and guidelines for optimizing accuracy and reducing impact on biosynthetic parameters are suggested. Adherence to certain analytic guidelines, particularly attention to concentration effects on mass isotopomer ratios and maximizing enrichments in the isotopomers of interest, reduces error. Improving instrument accuracy for quantification of isotopomer ratios is perhaps the highest priority for this field. In conclusion, MIDA remains the “equation for biosynthesis,” but attention to potentially confounding factors and analytic performance is required for optimal application.

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of 2 H2O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% 2 H2O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0 -200 g) to ovariectomized rats increased mammary epithelial cell proliferation, according to a doseresponse relationship up to the 100 g dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose-response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the 2 H2O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270 -400 days and die-away values after 2 H2O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1-1.5% new cells per day, whereas obese ad libitum-fed ob͞ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term 2 H2O enrichments in body water were achieved by daily 2 H2O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body 2 H2O enrichment (Ϸ3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with 2 H2O was 0.056 (CD4 ؉ ) and 0.043 (CD8 ؉ ) (replacement rate <0.1% per day). In summary, 2 H2O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells. deuterated water ͉ cell proliferation ͉ DNA synthesis ͉ adipogenesis ͉ vascular smooth muscle cell proliferation