Main finding of this study is that feto: maternal ratios were higher in hypertension and diabetes group compared to control group. Maternal diazepam concentrations were statistically higher in control and hypertension group compared to group of women with diabetes. Umbilical cord arterial: venous plasma concentration ratios were similar in all groups and they were not statistically different.
The strength of this study is a prospective design which did not disturb regular clinical procedures. Limitation of this study is smaller number of pregnant women in diabetes group than in control and hypertension group.
In a majority of previous studies diazepam umbilical cord plasma concentrations exceeded maternal concentrations [23–25], although feto: maternal ratios below 1 have also been reported . Diazepam is highly liposoluble and crosses placenta by passive diffusion via transcellular route [27, 28] thus diazepam transfer depends on properties of the placenta and is in a function of multiple factors such as placental blood flow, protein binding, lipid solubility and ionization constant (pKa).
In our study there were 76% of feto: maternal ratios above and 24% of feto-maternal ratios below 1. There were also feto-maternal ratios above 2 (16% control group; 57% hypertension group; 36% diabetes group). This study demonstrates that feto: maternal ratios in diabetes and hypertension groups were significantly higher than in control. However, there was no difference between the ratios of the diabetes and hypertension groups. Since the administered doses of diazepam were similar in all groups, our findings show that there is a difference in diazepam transfer across placenta in the diabetes and hypertension groups.
There is no drug-drug interactions observed or reported [29–32] between diazepam and other administered drugs/anaesthetics, which indicates that diazepam absorption studies were not compromised.
Since no known interaction on pharmacokinetic level are expected between diazepam and insulin it was not considered that insulin treatment in 3 women in diabetes group had effect on concentration data.
Interestingly, in one study, a single oral dose of 5 mg diazepam taken before dental treatment did not influence blood glucose level in nondiabetic and non-insulin dependent diabetic subjects . But diazepam may alter insulin secretion and insulin sensitivity after a single administration in healthy volunteers , and diazepam-induced hyperglycemia, might be related to changes in serum chromium levels . However in this study the increase in glycemia in diabetic women would be the result of the stress due to operation rather than diazepam treatment.
Binding of diazepam to human serum albumin can be influenced by hypertension and diabetes due to changes in hemodynamic properties or by drugs used for their treatment. Since diazepam is highly bound to proteins (96-99%), and protein bound fraction of drug do not cross placenta , its metabolism rate and transplacental passage depends on free drug fraction. Diazepam is more protein bound in fetal circulation than in maternal circulation and it has one binding site on human serum albumin . Since there is no known interactions at the level of protein binding between methyldopa or insulin nor drugs used in anaesthesia, observed higher feto: maternal ratios in diabetes and hypertension group could be consequence of the increased level of some endogenous substance in diabetes and hypertension, like free fatty acid which displace diazepam from proteins [38, 39].
In the study by Ridd et al.  were found differences in protein binding of diazepam in maternal and diazepam plasma but free diazepam fractions were similar on both sides of placenta. Unbound drug concentrations in plasma are responsible for the obtained pharmacological effects, thus it would be expected that partial displacement of diazepam from plasma proteins would result in the increase in the magnitude of its pharmacological effects [40–43]. In our study there were no side effects in newborns (i.e. Apgar scores had normal values) or in mothers but this does not imply that there were no increase in diazepam free fraction. Since total clearance of diazepam is directly proportional to free diazepam fraction [44–46] and increased free diazepam fraction would lead to higher elimination and disappearance of diazepam from blood. This could be proved through AUC values after delivery which were statistically higher in control group compared to hypertension and diabetes group that implies that elimination of diazepam from central compartment is higher in hypertension and diabetes group and that is likely that there were more unbound diazepam in the blood in these groups.
Also other mechanisms such as metabolism and active transport could lead to differences in feto: maternal unit. Diazepam is metabolized by CYP enzymes some of which are present in placenta like CYP3A4 . Presence of CYP 3A4 in human placenta is more than controversial, based on original and review articles , although some expression may be present at a very low, not functionally significant level. The most direct study on the absence of CYP3A-catalyzed metabolism was conducted on carbamazepine . Previous studies showed that diazepam is not substrate for P-glycoprotein  but recent study suggested that diazepam is a modifier of P-glycoprotein because it behaved as an activator of the P-glycoprotein ATP-ase activity . The potential of diazepam to modify P-glycoportein could affect placental transfer of other drugs which are substrates of P-glycoprotein. Still observed higher transplacental transition of diazepam in diabetes and hypertension group in our study may be due to lower activity of some other efflux proteins.
Ionic composition of cellular and extracellular space is altered in hypertension and diabetes due to altered work of some ion exchange pumps like Na+/H+ and Ca2+/H+[52, 53]. This could have influence on degree of diazepam ionization since if ionic strength of solution is lower dissociation balance shift to neutral molecules. Namely, diazepam is weak base (pKa 3.3), mainly unionized in blood (pH 7.4) when unionized molecules leave plasma balance is shifted to new ionization. Since fetal plasma is slightly more acidic, 0.1 lower than maternal plasma pH, the un-ionized free drug crossing the placenta becomes ionized and is 'trapped’ in the more acidic fetal circulation [54, 55]. In this study only in control group in all cases umbilical cord arterial: venous diazepam plasma concentration ratios were close to one, but in hypertension and diabetes group half of ratios were over one which means that higher maintenance of diazepam occurred within infants of mothers with diabetes or hypertension. As it was single dose use of diazepam and Apgar scores were not statistically different between groups there were no observed differences in pharmacological effect on neonates between groups. Some studies showed that maternal diazepam medication affects the beat-to-beat variability in the newborns  but duration of the effect is more profound in the chronic and infusion diazepam groups. However, it is known that diazepam free fraction increases shortly after birth which explains adverse effects observed clinically in some diazepam exposed neonates . Thus it could be concluded that hypertensive and diabetic group neonates were in higher risk of serious diazepam side effects.